JPH07322895A - Radical enhancer - Google Patents

Abstract

PURPOSE:To obtain the subject reagent comprising a p-hydroxyacetoanilide derivative used for increasing the optical density or radical signal strength of an oxidatively coloring reagent for determining a peroxidase, etc., and capable of highly sensitizing the measurement of the peroxidase, etc. CONSTITUTION:When a peroxidase and a substance having the same catalytical action as that of the peroxidase are measured using an oxidatively coloring reagent in the presence of hydrogen peroxide and the peroxidase or a labeled substance having the same catalytical action as that of the peroxidase, in a method for determining the peroxidase or the substance having the same catalytical action as that of the peroxidase, a p-hydroxyacetoanilide derivative of the formula (R is a 1-5C alkyl, an alkoxyl) (e.g. 4-acetoamidephenol) is used for increasing the optical density or radical signal strength of the used oxidatively coloring reagent (e.g. o-phenylenediamine), and a tartaric acid and/or sodium tartrate buffer solution is used as a substrate buffer solution to determine the peroxidase or the substance having the same catalytic action as that of the peroxidase.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a peroxidase (hereinafter
(Abbreviated as POD) and a substance having a catalytic action equivalent thereto, particularly, using POD and a catalyst equivalent to that using an oxidative coloring reagent in the presence of hydrogen peroxide and a labeled substance having a catalytic action equivalent to POD. The present invention relates to a method for measuring a substance having an action.

[0002]

2. Description of the Related Art In general, a clinical test method using an enzyme reagent or an antibody is an assay method having extremely high selectivity due to the substrate specificity of the enzyme or the antigen specificity of the antibody, and is affected by a complicated biological matrix. It is an important method because various biological components can be easily quantified without it. Of the enzymes, POD and alkaline phosphatase (hereinafter abbreviated as ALP) are widely used as labeling agents for various proteins, and antigens, antibodies, etc. are labeled with such POD and alkaline phosphatase, and the target compound ( antigen,
The target compound (antigen, antibody, etc.) can be quantified with high sensitivity by using an oxidative coloring system or an enzyme substrate after acting on the antibody, etc.).

As a POD measuring reagent which has been used conventionally,
Oxidative color reagents such as o-phenylenediamine (hereinafter abbreviated as OPD) are generally used, and POD or hydrogen peroxide is detected by oxidative condensation in the presence of hydrogen peroxide and measuring the condensate. Oxidative color reagents other than OPD include phenol, N, N, -dimethylaniline, 4-chlorophenol, N-ethyl-N-sulfopropylaniline (hereinafter abbreviated as ALPS), N-ethyl-N- (2-hydroxy). -3-Sulfopropyl) -m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (hereinafter abbreviated as ALOS), N- (3-sulfopropyl) aniline (hereinafter abbreviated as HALPS) , 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (hereinafter abbreviated as ABTS) and the like are used.

In recent years, the chemiluminescence method has been attracting attention due to its high sensitivity, which can be achieved by an enhancer. For example, in the case of luminol, the emission is said to be about 500 times stronger by adding p-iodophenol (Thorpe, GHG and Kricka, LJ: Metho.
ds in enzymol., 133 , 331, 1985). Moreover, 3- (2'-spiroadamantane)-which is a substrate of alkaline phosphatase
4-Methoxy-4- (3 "-phosphoryloxy) phenyl-1.2-
Dioxetane 〔3- (2'-spiroadamantane) -4-metoxy-4-
(3 "-phosophoryloxy) phenyl-1.2-dioxetane] (AMPPD
), A remarkable enhancement of light emission is recognized in the coexistence of a quaternary amine polymer.

[0005]

Although various enhancers (enhancing agents) have been reported for the chemiluminescence method as described above, reports of enhancers for chromogenic substrates are extremely rare. JP-B-51-37555 discloses that color tone changes depending on glucose concentration by simultaneously adding o-aminophenol, p-aminosalicylic acid, naphthylamine, etc. to ABTS, but high sensitivity There is no description about conversion.

An object of the present invention is to improve the measurement sensitivity of POD or a substance having a catalytic action equivalent to that of POD by an oxidative coloring reagent.

[0007]

[Means for Solving the Problems] As a result of various studies for solving the above problems, it has been noted that the chromogens such as ABTS, ALPS, HALPS, and ALOS that have been conventionally used as chromogenic substrates for POD are radicals. , The sensitivity or S / N ratio of the measurement system could be improved by adding the second substrate at the same time.

That is, the present invention provides the following general formula (1) used for the purpose of enhancing the optical density or the signal intensity of radicals of an oxidative coloring reagent used in a method for quantifying a peroxidase or a substance having a catalytic action equivalent to that. Is a radical enhancer composed of a p-hydroxyacetanilide derivative represented by:

[0009]

[Chemical 3] In the formula, R is an alkyl group or an alkoxyl group, and the carbon number thereof is 1 to 5, preferably 1 to 3.

In the present invention, the enhancer means an enhancer for increasing the number of photons and fluorescence intensity in emission analysis,
A radical enhancer is a second substrate that acts to increase or increase the radical intensity or absorbance of the main substrate by the action of the second substrate that has been radicalized by the action of POD on the main substrate and the second substrate. Is called a radical enhancer or simply an enhancer.

(Synthesis of Compound) The compound represented by the above general formula (1) used in the present invention can be synthesized using a compound having p-hydroxyaniline as a basic skeleton as a starting material. For example, 4-acetamidophenol (R ¹ : CH ₃ , compound A) can be synthesized by reacting 4-aminophenol with acetic anhydride.
-4-propionylaminophenol (R ¹ : C ₂ H ₅ , compound B) can be synthesized by reacting 3-methyl-4-aminophenol with propionic anhydride, and by recrystallization from water and treatment with activated carbon. Refined.

In addition to these, typical compounds of the compound (1) include those in which R is propyl (compound C), methoxy (compound D) and ethoxy (compound E), respectively. These representative compounds AE are shown in FIG.

(Peroxidase) In addition to the substances containing iron porphyrin protein such as peroxidase obtained from horseradish, the peroxidase to be measured in the present invention and the substance having a catalytic action equivalent to that, Super Oxid
e Dismutase, potassium ferricyanide, oxidizing agents such as sodium metaperiodate, ferric iron compounds, etc.

(Oxidative coloring reagent) ABTS, ALPS, HA described above
Radical color formers such as LPS and ALOS are used. Such a radical-forming color former is generally a substrate for peroxidase, has a benzene ring, and the ortho-position of the benzene ring is not occupied by other substituents, and is a substrate for radicalization.

(Substrate buffer) In the measurement using the radical enhancer of the present invention, the substrate buffer used is arbitrary, but the effect of the enhancer is influenced by the type. For example, when ABTS is used as the main color-developing agent, as shown in FIG. 2, the coloration without the enhancer is stronger when the citrate / phosphate buffer is used as the substrate buffer, but the compound A is used as the enhancer. The enhancement effect when you are tartaric acid
The sodium tartrate buffer is more pronounced.

Further, ABTS radicalizes over time due to the effect of hydrogen peroxide in the substrate buffer, which causes an increase in the background value, which greatly affects the sensitivity and reproducibility at low values, but is shown in FIG. Thus, in the absence of peroxidase, the tartaric acid buffer had a background value of ABTS for various enhancers of the present invention as compared with the background value (peak height value 43 mm) in the citrate / phosphate buffer solution. Suppress the rise of.

[0017]

The p-hydroxyacetanilide derivative of the present invention is also used as a fluorescent reagent in the reaction between hydrogen peroxide and peroxidase. The mechanism of the action of the oxidative coloring reagent as an enhancer in the present invention is not always clear. Although it does not exist, it is considered to have a different mechanism of action as a fluorescent agent.

[0018]

EXAMPLES Next, the POD quantification method using various p-hydroxyacetanilide derivatives will be described with reference to Examples, but the present invention is not limited by the following Examples as long as the gist thereof is satisfied. .

The following reagents were used in the following examples.

Hydrogen peroxide: 30% hydrogen peroxide solution manufactured by Kanto Chemical Co., Inc.

HRP: horseradish peroxidase (activity 250 units / mg, Wako Pure Chemical Industries, Ltd.) was added to the enzyme dilution solution (0.2% bovine serum albumin, SIGMA) to adjust the activity.
Adjusted to 10 ^-3 units / ml and 10 ^-4 units / ml.

Main substrates: ABTS (manufactured by Nippon Kayaku Co., Ltd.), ALPS (manufactured by Dojindo Laboratories), HALPS (manufactured by Dojindo Laboratories), ALOS (manufactured by Dojindo Laboratories) all in ultrapure water at 3.0. Adjust to a concentration of mg / ml.

Enhancer solution: The above compounds A to E were dissolved in ultrapure water by heating to adjust the concentration to 3.0 mg / ml.

Substrate buffer-1: Citrate / phosphate buffer.
Disodium hydrogen phosphate dodecahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.
Prepared with 208 g, citric acid (Wako Pure Chemical Industries, Ltd.) 0.281 g, 30% hydrogen peroxide water 0.015 ml and ultrapure water 30 ml. pH: 4.0.

Substrate buffer-2: Tartrate buffer. After mixing 0.1M tartaric acid (Kanto Chemical Co., Inc.) and 0.1M sodium tartarate dihydrate (Kanto Chemical Co., Inc.) to adjust the pH to 4.4, add 30% hydrogen peroxide solution to a final concentration of 0.015%. Added to.

Examples 1 to 5, Comparative Example 1 (Enhancer effect of POD standard solution) 70 μl of ultrapure water,
Enhancer (A to E) 30 μl each, Substrate buffer-1
Alternatively, -2 100 μl and main substrate ABTS 50 μl are mixed, and HRP 50 μl with an activity of 10 ⁻⁴ units / ml is added at room temperature (22-24
Electron spin resonance (E
Table 1 shows the results of measuring the signal intensity of SR) with a gain of 1 × 100.

The ESR was measured using FR80 manufactured by JEOL Ltd., and the signal intensity is shown in peak height (mm) under the following measurement conditions.

Magnetic field: 3356 ± 250 G, magnetic field modulation: 100 kHz 10 G, power: 20 mW, gain: 1 × 100, response: 1.0 sec.

[0029]

[Table 1] In addition, among various enhancers, the ESR spectrum of Compound A having a large amplification effect is shown in FIG.

Examples 6 to 10 and Comparative Examples 2 to ³ Immunosupplied by Nippon Intermed Co., Ltd. in the same manner as the main substrate ABTS except that the main substrate was ALPS and the activity of HRP was 10 ^-3 units / ml. Table 2 shows the results of measuring the absorbance at a maximum absorption wavelength of 450 nm using Reader NJ-2000.

[0031]

[Table 2] Examples 11 to 20 and Comparative Examples 4 to 7 For the cases where the main substrates are HALPS and ALOS, the results of measuring the absorbance at the maximum absorption wavelength in the same manner as in the case of the main substrate ALPS are shown in Table 3 (HALPS, maximum absorption). The wavelength is 680 nm) and Table 4 (ALOS, maximum absorption wavelength 450 nm).

[0032]

[Table 3]

[0033]

[Table 4] Examples 21 to 26 (Improvement of S / N ratio) 70 μl of ultrapure water, 30 μl of each of Compounds A to E, 100 μl of substrate buffer-2 (tartrate buffer), AB
Mix 50 μl of TS and 50 μl of ultrapure water and let stand at room temperature.
The blank value after incubation for a minute was measured by ESR.

The measurement results are shown in FIG. Here, the control indicates the measured value in the absence of the enhancer. The measurement conditions are as follows.

Magnetic field: 3356 ± 250 G, magnetic field modulation: 100 kHz 10 G, power: 20 mW, gain: 6.3 × 100, response: 1.0 sec.

The background value of ABTS (corresponding to the control in FIG. 3) of the substrate buffer-1 was measured, and the peak height value was 43 mm.

On the other hand, the activity of 10 ^-4 was used instead of 50 μl of ultrapure water.
The signal intensity of electron spin resonance (ESR) was measured after adding 50 μl of units / ml of HRP and incubating at room temperature for 15 minutes. (See FIG. 2) As shown in FIG. 3, the substrate buffer solution-2 has a control value
The value is 10 mm, and the blank value hardly changes even by adding the compounds A to E, and suppresses the increase in the background value (RB) of ABTS to about 1/4 as compared with the substrate buffer-1.

However, since the signal intensity of ABTS in the HRP-added system is suppressed to about 1/2 of that in the case of the substrate buffer-1 (see Comparative Example 1 in Table 1), the S / N value is about Only double the improvement. On the other hand, as shown in Table 1, when the compounds A to E are added using the substrate buffer solution-2, the signal intensity is enhanced 3.4 to 6.1 times as compared with the system without addition (Comparative Example). This is an improvement in the amplification rate of 1.6 to 2.6 times compared with the case of using the substrate buffer-1, and finally an improvement in the S / N ratio of 3.2 to 5.2 times is possible.

[0039]

[Effects of the Invention] When the main substrate is radicalized by oxidation by peroxidase and a substance having a catalytic action equivalent to that, by adding the p-hydroxyacetanilide derivative of the present invention, its ESR signal or absorbance is significantly enhanced, By selecting the substrate buffer used, the background of the measurement system can be lowered and the detection sensitivity can be improved.

[Brief description of drawings]

FIG. 1 shows the structural formulas of representative compounds A to E of the present invention.

FIG. 2 shows an enhancer enhancing effect of a substrate buffer.

FIG. 3 shows the changes caused by the background of substrate buffer-2 and its enhancer.

Claims (3)

[Claims] 1. A method for quantifying peroxidase or a substance having a catalytic action equivalent to that of peroxidase, which is represented by the following general formula (1), which is used for the purpose of enhancing optical density or radical signal intensity of an oxidative coloring reagent used.
A radical enhancer composed of a p-hydroxyacetanilide derivative. [Chemical 1] (In the formula, R is an alkyl group or an alkoxyl group having 1 to 5 carbon atoms.) 2. A method for quantifying peroxidase or a substance having a catalytic action equivalent thereto, which comprises using POD and its equivalent using an oxidative coloring reagent in the presence of hydrogen peroxide and a labeling substance having a catalytic action equivalent to peroxidase. A method for quantifying a peroxidase or a substance having a catalytic action equivalent to that of a peroxidase, characterized by using a p-hydroxyacetanilide derivative represented by the following general formula (1) as a radical enhancer when measuring a substance having a catalytic action. [Chemical 2] (In the formula, R is an alkyl group or an alkoxyl group having 1 to 5 carbon atoms.) 3. A method for quantifying peroxidase or a substance having a catalytic action equivalent to that of POD and its equivalent using an oxidative coloring reagent in the presence of hydrogen peroxide and a labeling substance having a catalytic action equivalent to peroxidase. The method according to claim 2, wherein a tartaric acid / sodium tartrate buffer is used as a substrate buffer when measuring a substance having a catalytic action.