JPH03285697A - Method for testing chemoluminescence - Google Patents

Abstract

PURPOSE:To measure a peroxy lipid, etc., in high sensitivity by reacting a peroxidase, an oxidizing agent and a luminous substrate selected from luminol and isoluminol with each other and subsequently detecting a component relating to the produced chemoluminescence. CONSTITUTION:A peroxidase, an oxidizing agent and a luminous substrate selected from luminol and isoluminol are reacted with each other in the presence of a chemoluminescence sensitizer to generate the chemoluminescence, thereby detecting or quantifying one of the reagents relating to the reaction. The sensitizer includes hydrazobenzene, indigo, 3-methyl-2-benzothiazolinone hydrazone.HCl, alpha-naphthoquinone, indophenol, phenylhydrazine, 8-hydroxyquinoline, phenolphthalein, L-tryptophan and N,N-dimethylindoaniline.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application 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 management or the early detection of abnormal conditions in the body has been recognized, and the importance of early detection of abnormal conditions in the body has been recognized.
As a means of understanding abnormalities, simple and rapid quantitative analysis methods for biological components that serve as biochemical indicators are being developed and applied, particularly methods using enzymatic methods with excellent specific selectivity. At that time, biological components serving as biochemical indicators are converted into measurable signal compounds (eg, hydrogen peroxide, NAD (P) +, etc.) using selective enzymes, and then quantified. Hydrogen peroxide is particularly frequently used as such a final signal compound, and the development of an enzyme-coupled reaction system that provides hydrogen peroxide is also being carried out in an elaborate manner. To quantify hydrogen peroxide, the final signal compound,
Chemiluminescence reactions using peroxidase (hereinafter abbreviated as POD) as a catalytic enzyme are widely used. The present invention relates to a method for enhancing and stabilizing luminescence in this chemiluminescent reaction.

(Prior Art) The chemiluminescent reaction by POD, using hydrogen peroxide as an oxidizing agent, is expressed by the following reaction formula: 0D H20□+AH2-→ 2H,, O+A( In the formula, Al1
represents the reactive substrate of POD) This reaction can lead to a quantifiable luminescence (emission of photons). There are also known methods in which A produced in the reaction equation is a dye or fluorescent substance, but the method that measures the amount of luminescence has the highest sensitivity, so it is possible to reduce the amount of biological samples, such as the amount of blood collected during periodic diagnosis. This method has various advantages, such as being able to sufficiently dilute the biological sample before carrying out the reaction, thereby reducing the influence of reaction-interfering substances in the sample.

In addition, the high measurement sensitivity of the method of measuring luminescence amount will make it possible to measure biological components that could not be used as biochemical indicators because they did not reach the measurement range. It is expected.

Although the detailed mechanism of chemiluminescence caused by POD has not been completely elucidated, studies using peroxidase derived from horseradish (hereinafter abbreviated as HRP) have been widely conducted. However, although HRP has the advantage of emitting light under milder conditions than other inorganic light-emitting catalysts, the light-emitting catalytic ability of HRP is insufficient for use in microquantitative reactions of biological components. As a means of solving this problem, various compounds that enhance the chemiluminescence of HRP have been found (Japanese Patent Application Laid-Open No. 171839/1983 and Japanese Patent Application Publication No. 59/500252), but such sensitizers Even with the use of HRP, the chemiluminescent catalytic ability of HRP has not yet reached a satisfactory level.

Furthermore, available HR,P is often a mixture of multiple isozymes, which affects the accuracy and reproducibility of quantitative reactions, and HRP has complex luminescence reaction behavior, such as luminescence reaction rate. There is also the problem that it is difficult to control the reaction when performing quantitative measurements, such as by exhibiting a pulsating luminescence pattern that fluctuates over time.

Therefore, the present inventors attempted to search for POD with superior luminescent catalytic ability than HRP, and discovered peroxidases (hereinafter abbreviated as ARP and Ccp) produced by fungi belonging to the genus Arthromyces and Coprinus, and discovered that It is disclosed in Publication No. 61-043987. 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 (Japanese Patent Application Laid-Open No. 63-21939).
Publication No. 8).

(Problems to be Solved by the Invention) The present invention utilizes the excellent luminescent catalytic ability of ARP or CCP,
It is an object of the present invention to provide a chemiluminescence quantitative method that is further enhanced using a sensitizer and made more suitable for quantifying trace amounts of biological components and the like.

The present invention also aims to increase the catalytic ability of these enzymes by enhancing the catalytic ability of these enzymes under conditions where the ratio of light emission (signal) generated by ARP or CCP to non-specific light emission (noise), that is, the signal/noise ratio is high. The object is to provide a chemiluminescence assay particularly suitable for the determination of components.

Moreover, since the sensitized chemiluminescence reaction often stops after a short time, the present invention overcomes these drawbacks and allows the enhanced luminescence catalyzed by ARP or CCP to last as long as possible, making it simpler and easier to use. The purpose of the present invention is to provide a chemiluminescence quantitative method that enables quantitative determination using a standardized analyzer.

(Means for Solving the Problems) For the above purpose, the present inventors have conducted various studies and found that hydrazobenzene, indigo, 3-methyl- 2-
Benzothiazolinone hydrazone/HCI, α-naphthoquinone, indophenol, phenylhydrazine, 8-
Hydroxyquinoline, phenolphthaline, L-tryptophan, N,N-dimethylindoaniline, calcein, indigo carmine and resorcin were found.

Among these sensitizers, hydrazobenzene, indigo and 3-methyl-2-benzothiazolinone hydrazine HCI are notable for their long duration of enhanced chemiluminescence and high signal-to-noise ratio. It is particularly preferable.

Therefore, 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 method is characterized by producing chemiluminescence in the presence of a sensitizing agent.

The peroxidase used in the present invention is peroxidase derived from the genus Arthromyces (ARP) or peroxidase derived from a fungus of the genus Coprinus (ccp).

An example thereof is described in Japanese Patent Application Laid-Open No. 61-043987. Peroxidases also include those produced by genetic engineering. Note that horseradish-derived peroxidase (HRP) is not used in the present invention.

As an example of carrying out the method of the present invention, the following conditions can be set: a) ARP or CCP 0.01 μg to 1000 μg/1) Oxidizing agent 0.01 nmol to 100 mmol/1 c) Luminol or isoluminol 0 , 5 μmol −
200 mmol/(in) chemiluminescent reaction sensitizer 1 μmol/1 to 100 mmol/1 e) pH 8 to 11 to) Temperature 10 to 60°C At least one of the main reaction components of the above a) to 2) to be measured. A mixed solution is prepared and placed in a reaction tank (for example, a test tube). Next, the missing main reaction component is added to the reaction vessel to initiate the chemiluminescent reaction. The light produced by the reaction is quantified by standard measurement equipment, such as a photomultiplier tube, and the signal is sent to a recorder, oscilloscope or scalar for display or recording.

Alternatively, if desired, the reaction may be observed with the naked eye, or the reaction may be quantified by reacting on a photographic plate. The present inventors measured the amount of luminescence using a luminometer (Lumac/3M
Biocounter M2O10 type, manufactured by 3M Pharmaceutical Co., Ltd.)
Measured using

According to the method of the present invention, oxidizing agents, luminescent substrates, peroxidases, and chemiluminescent sensitizers can all be detected and quantified. biological peroxides) are important targets for measurement.

When quantifying components such as proteins, hormones, steroids, nucleic acids, and metabolic intermediates in biological samples using enzyme-linked immunosorbent methods, it is possible to quantify components such as proteins, hormones, steroids, nucleic acids, and metabolic intermediates in biological samples by directly binding peroxidase to the antibody to be measured and adding hydrogen peroxide; It may be hydrogen peroxide generated in the reaction system by the action of an enzyme (coupled enzyme) such as alkaline phosphatase or glucose oxidase bound to an antibody, and based on the result, the original amount of the component in the biological sample can be determined. can be known. The method of the present invention can be used for various analytical purposes other than trace quantification of biological components for analysis items for which hydrogen peroxide produced directly or as a signal can be used as an indicator.

EXAMPLES 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 the Examples.

Example 1 Increasing the amount of light using ARP RZ=2. purified by the method described in JP-043987. I used one from O (RZ is A40
3 to A205). Luminol and hydrogen peroxide were purchased from Nacalai Tesque. Chemiluminescent reaction sensitizers were purchased from Subalai Tesque, Tokyo Kasei Kogyo, Sigma, or Aldočić. Other reagents were purchased from Nacalai Tesque.

For the reaction, 20 μg/j2 of ARP and 1 m
A solution of luminol of mole/Il was prepared, and 650 μm of this solution was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 061 to 1 mM in a disposable plastic test tube. After mixing, the reaction was started by placing the luminometer at the luminescence measurement site of the luminometer and adding 100 μm of 0.15 mM hydrogen peroxide prepared with the above buffer solution. The reaction temperature was 25°C. Luminescence was recorded using an average luminescence integrator built into the luminometer (in this example, the results are shown as an average cumulative amount of luminescence over 30 seconds) and an external output signal from the luminometer using a recorder. The observed average luminescence amount and signal/noise ratio (S/N) are shown in Table 1.

Table 1 Addition concentration (mM) Luminescence amount 179 0.1 34986 1.0 256604 0.1 36702 1.0 168860 0.1 176878 1.0 52649 0.1 175027 1.0 235182 0.1 30489 1.0 66859 0.1 275834 1.0 80960 8-hydroxyquinoline 1.0 42462L-tryptophan 1.0 15684N,N-dimethylindoaniline 0.1 320
27 Calcein 1.0 2262 Findigo Carmine 1.0 33482 Resorcin
1.0 25083 and Dracin MCl 3-Methyl-2-benzothiazolinone No additive sensitizer Phenylhydrazine Indigo Indophenol α-Naphthoquinone Hydrazobenzene S/N 39 77 751 76 73 422 316 64 1 082 572 95 0 831 54 464 11 97 672 For the reaction, 1 μg and 5 μg/l of ARP,
A solution containing 1 mmole/It of luminol was prepared, and 650 μm of this solution was dissolved in DMSO to a concentration of 0.1 to 1 mM in a disposable plastic test tube.
After mixing with Example 1, the same procedure as in Example 1 was carried out. The reaction was followed for 3 minutes after the start of the reaction, and the durability of the luminescence reaction was compared as the cumulative amount of light every 30 seconds. The results are shown in Table 2.

Example 3 Sensitizer p-hydrogen 1-hong.

AR was measured under the same measurement conditions as Example 1 except that the reaction was started by adding 1 mM hydrogen peroxide
The effect of the concentration of hydrazoene, which is one of the sensitizers of the present invention that undergoes a chemiluminescent reaction using P as a catalyst, was investigated. As a result of measurement as the average cumulative amount of luminescence for 60 seconds,
The maximum sensitizing effect was obtained at 0.5-1 mM (30-60 μM final concentration). The results are shown in Figure 1.

Example 4 Effect of pH on the sensitized rapid luminescence reaction when 71 mM of hydrazobenze, one of the sensitizers of the present invention, was added. , under the same conditions as in Example 3, compared with the case of I) MSO without sensitizer (control).

In order to investigate the effect of pH, 50mM1・Squirrel・11C1
Buffer (pH 8.3-9.5), 50 mM birophosphate/HCI buffer (pH 8.7 to 9.6), and 100 mM glycine/NaOH buffer (pH 9.0-11.0) were used. As a result, it was found that the optimum pH shifted to the neutral side compared to the case without the addition of hydrazobenzene, and the luminescent reaction reached its maximum at around pH 9.6 of the pyrophosphoric acid/HCI buffer. The results are shown in Figure 2. In Figure 2, the chemiluminescence sensitized with hydrazobenzene is shown as a solid line, and the chemiluminescence without the addition of hydrazobenzene is shown as a broken line (2, white circles are).
- Chemiluminescence in Lis-HCI buffer, black circles in pyrophosphate-HCI buffer, and double circles in glycine-NaOH buffer.

Example 5 Sensitization of ARP Quantifiable by light Using hydrazobenzene, which is one of the sensitizers of the present invention, the quantifiable range of ARP was investigated under the conditions shown below based on the sensitized luminescent reaction. At the same time, HRP (RZ=3.5, manufactured by Toyobo Co., Ltd.)
Chemiluminescence, in particular, a sensitized luminescence system for HRP using 6-hydroxybenzothiazole, which is already known as a sensitizer for HRP (Thorpe et al., Anal.

Biochem, 145 96.1985).

The reaction was carried out in advance with 50mM birophosphate/HCI buffer (pH 9.6) for ARP, and O,IM Tris/HCI buffer (pH 8.5) for HRP.
) to 2 μg to 4 mg/I! (The final concentration of the reaction solution is TO
, i6μg-325, czg/ff1. m equivalent) (7
) POD, an effect of 1 mmole/l luminol was prepared and 650 μm of this was mixed in a disposable plastic test tube with 50 μm of the above sensitizer or control DMSo dissolved to 1 mM in DMSO, The reaction was started by adding 100 μm of hydrogen peroxide. Other reaction conditions were the same as in Example 1. A with hydrazobenzene added as a sensitizer
It was found that in the RP-sensitized luminescence system, quantification is possible in a lower concentration range and the quantifiable range is broader than in the HRP-sensitized luminescence system (0.16 μg as the final reaction ARP concentration). ~16.3μg/j2
was quantifiable). The results are shown in Figure 3.

In Figure 3, white circles are ARP (hydrazobenzene added), circles with x are ARP (no sensitizer added), black circles are HRP (6-hydroxybenzothiazole added),
The double circles indicate the quantifiable range of HRP (no sensitizer added).

Example 6 Sensitization of ARP Photoinduced hydration The concentration of ARP was 2.5 mg/1, and the amount of hydrogen peroxide added to initiate the reaction was 1 nM to 4. The quantifiable range of hydrogen peroxide by the method of the present invention was investigated under the same conditions as in Example 5 except that the concentration was set to mM. In the ARP-sensitized luminescence system in which hydrazobenzene was added as a sensitizer, hydrogen peroxide at a final reaction concentration of 0.25 to 500 nM could be quantified. The results are shown in Figure 4.

Example 7 Increasing the amount of CCP In order to confirm that the chemiluminescence of CCP is increased by the sensitizer of the present invention in the same way as ARP, CCP was added instead of AR, P.
A test similar to that in Example 1 was conducted using the following. CCP is RZ
2.1 was used. As a result, the sensitizer of the present invention showed the same sensitizing effect in CCP as in ARP.

The results are shown in Table 3.

Table 3 Sensitizer added concentration Luminescence amount S/N No additive hydrazobenzene indigo MBTH* 0.1mM 1.0mM 0.1mM 1.0mM 0.1mM 194 1639 3263 8019 15935 10643 93 940 771 72 55 9149 Reaction is , 1 μg/l of CCP and 0.0 μg/l of sensitizer.
The same procedure as in Example 2 was performed except that the solution was dissolved in DMSO to a concentration of 1 mM. As a result, compounds that showed a sustained luminescence reaction in ARP showed similar effects when placed in CCP. The results are shown in Table 4.

9 0 Example 9 A certain amount once to K NADH was quantified by the method of the present invention.

Hydrazobenzene was used as a luminescence sensitizer.

Prepare 50mM birophosphate/HCI buffer (pH 9) in advance.
, 3) with ARPo, 1/1 and Luminol 1 mm
600 μm of a solution containing ole/Il, 0.1 mM to 1 mM in the above buffer (final concentration of 6°25 μM to
50μ of NADH solution dissolved to give 62.5μM)
11 and 1 mM hydrazobenzene/DMSO solution 50
Add 100 μm of 1.6 μM 1-methoxy 5-methylzenadinium methyl sulfate (
The luminescence reaction was initiated by adding the above buffer solution).

The method for measuring luminescence was the same as in Example 1, except that the average luminescence amount was measured for 60 seconds. The results are shown in Figure 5.

Example 10 Amount of Cumene Hydroperoxide Cumene hydroperoxide, which is a peroxide, was quantified as an oxidizing agent in a chemiluminescent reaction using ARP. Hydrazobenzene was used as a sensitizer. ARP 0.4
50mM pyrophosphate HCI at a concentration of mg~10■/l
1mM cumene hydroperoxide solution was dissolved in a buffer solution (pH 9°3) and 100% of cumene hydroperoxide solution was added instead of hydrogen peroxide.
Luminescence measurement was performed in the same manner as in Example 1, except that the reaction was started by adding μl. Table 5 shows the results of hydrazobenzene-sensitized luminescence obtained as the average luminescence amount for 60 seconds.

Table 5 0 4 82 11951.0
112 44322.0
164 86074.0 168
1169710 0 201
15328 Example 1 - 1 The reaction was carried out under the same conditions as in Example 1, except that the concentration of ARP was 2 μg/ff, and the external output signal from the luminometer was recorded. The sensitized luminescence pattern due to the addition of hydrazobenzene, which is one of the sensitizers, was investigated. The results are shown in Figure 6.

As is clear from this figure, the chemiluminescence enhanced by the sensitizer persisted stably for 10 minutes or longer.

Example 12 Sensitization by Vmax and Km Chemiluminescent reaction was carried out under the same conditions as in Example 5, except that the concentration of ARP or HRP was 100 μg/1 and the hydrogen peroxide added to initiate the reaction was 1 μM to 1 mM. I did it. Based on the amount of luminescence obtained at each concentration of hydrogen peroxide, Lineweaver φ Bark (L i
The Michaelis constant (Km) and the maximum velocity (vmax) were determined from the plot (double reciprocal plot) of new ave r - Burk). As a result, it was found that addition of the chemiluminescent sensitizer of the present invention hardly changes the Michaelis constant, but significantly increases the maximum velocity (Vmax). The results are shown in Table 6.

Table 6 Vmax (per 10-”'M POD) RP 00 ARP + Hydrasohenzene 150 Benzothiazole (Effects of the Invention) According to the present invention, the chemiluminescence catalytic ability is higher than that of HRP.
By using chemiluminescent sensitizers that further enhance the catalytic abilities of ARP and CCP, extremely sensitive methods for measuring hydrogen peroxide, lipid peroxide, etc. have become possible. The method of the present invention allows measurements to be performed under conditions with a high signal/noise ratio. In addition, the method of the present invention has excellent measurement accuracy because the sensitized chemiluminescence stably lasts for a long time, and is very suitable for analysis of biological components in clinical tests and the like.

[Brief explanation of the drawing]

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 results of quantifying the amount of hydrogen peroxide using the method of the present invention. FIG. 5 is a graph showing the results of quantifying NADH using the method of the present invention. FIG. 6 is a graph showing the persistence of chemiluminescence in the presence of a sensitizer.

Claims (1)

[Claims] 1. Reacting peroxidase; oxidizing agent; luminescent substrate selected from the group consisting of luminol and isoluminol to produce chemiluminescence, and detecting or quantifying any of the components involved in the reaction. In the method of
- A method characterized in that chemiluminescence is produced in the presence of a chemiluminescence sensitizer selected from the group consisting of dimethylindoaniline, calcein, indigo carmine and resorcinol. 2. The method of claim 1, wherein the sensitizer is selected from the group consisting of hydrazobenzene, indigo, 3-methyl-2-benzothiazolinone hydrazone.HC1. 3. The method according to claim 1 or 2, wherein the peroxidase is an enzyme derived from a fungus belonging to the genus Arthromyces or the genus Coprinus. 4. The component to be detected or quantified is hydrogen peroxide as an oxidizing agent, and the hydrogen peroxide is produced from another substance by the action of an enzyme, according to any one of claims 1 to 3. the method of.