JP2738482B2 - Enzymatic assay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzymatic assay useful for analyzing biological components and various food components. More specifically, the present invention relates to an enzymatic assay using a signal amplification system associated with redox cycling of nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) or its reduced form (hereinafter abbreviated as NADH). Things.

[0002]

2. Description of the Related Art NAD and its reduced form, NADH
Is a coenzyme with the highest biocontent and is involved in the oxidation-reduction reactions of many dehydrogenases by changing reversibly through the transfer of hydrogen atoms. These NAD and N
ADH has an absorption maximum at around 260 nm and 340 nm, respectively, and its absorbance change has conventionally been applied to the measurement of various enzyme activities. Apart from this, NAD or NADH is detected with high sensitivity. As a measurement method, a signal amplification system by enzymatic cycling is known (C. Bernofsky et al., Analytical Biochemistry
52, 452-458, 1973: CHS
elf et al., Clinical Acta, 148, 119-12
4, 1985).

[0003] In this assay, a signal that develops a color following the redox reaction of NAD or NADH is amplified by repeating the reaction (enzymatic cycling),
This is to detect the presence of D or NADH with high sensitivity. That is, as shown in the reaction formula below,
NAD acts as a coenzyme in the oxidation reaction of ethanol to acetaldehyde by ADH in the presence of alcohol dehydrogenase (hereinafter abbreviated as ADH) and its substrate, ethanol, and reduces it to NADH. Furthermore, this NADH is oxidized again to NAD in the presence of a tetrazolium dye and its reducing enzyme diaphorase,
At the same time, the tetrazolium-based dye is reduced to form formazan, and a coloring signal is obtained. Since NAD repeats redox under such conditions, the signal intensity gradually increases. When NADH is present in a sample, it develops color according to the same principle and amplifies its intensity.

[0004]

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[0005] NAD or NADH by such a system
Can be detected by measuring the activity of phosphatase (CHSelf et al., Supra) and the activity of NAD synthetase (Hideo Misaki, BIO INDUSTRY, Vol. 7, pp. 775-787, 19).
In particular, in the measurement of alkaline phosphatase, the detection sensitivity equivalent to that of the fluorescence or colorimetric method can be obtained despite the colorimetric method. (Ishikawa et al., “Immunoassay Method 3rd Edition”, 58-60)
P., Published by Medical Shoin).

[0006]

Conventionally, in a measuring system using such enzymatic cycling, ADH derived from baker's yeast has been used. However, the baker's yeast ADH, because its activity duration is short, had to add a large amount of ADH to the measurement reagent for repeatedly redox the NAD or NADH obtain sufficient color signals .

The present invention has been made in view of the above circumstances, and has solved the disadvantages of the conventional measuring method.
It is an object of the present invention to provide a new enzymatic assay capable of detecting AD or NADH with higher sensitivity.

[0008]

Means for Solving the Problems The present invention has been achieved to solve the above-mentioned problems by providing a catalyst for enzymatic cycling in a signal amplification system using nicotinamide adenine dinucleotide or its reduced form. The present invention provides an enzymatic assay using an alcohol dehydrogenase derived from Zymomonas sp.

Hereinafter, the structure and preferred embodiments of the present invention will be described in more detail. The detection of NAD and NADH in the enzymatic assay method of the present invention is performed by using a sample containing any one of them and ADH, diaphorase,
Alcohol, tetrazolium dye, surfactant, buffer, etc. are mixed to make a measurement solution, and tetrazolium dye
The change in the absorbance of formazan caused by the reduction may be measured.

In the present invention, A to be added to the measurement solution
As DH, those derived from Zymomonas genus are used, and ADH derived from Zymomonas mobilis is particularly preferable.
Zymomonas, a microorganism, can be obtained from the Fermentation Research Institute, American Type Culture Collection (ATCC), or the like. After culturing the obtained Zymomonas, ADH
Can be obtained to obtain ADH. The cultivation of Zymomonas can be performed in the same manner as general bacteria, and the purification can be performed in the same manner as general proteins. Preferably, European Journal of Biochemistry, Vol. 154, pp. 119-124 (1)
986). Further, the method described in the literature may be used for purification of ADH. ADH during measurement
The concentration is 0.01 to 1000 units / ml, more preferably 0.1 to 100 units / ml. One unit of the activity of ADH is defined as the amount of enzyme that oxidizes 1 μmol of ethanol per minute at pH 8, 30 ° C.

As the above components other than ADH in the measurement solution, the same components as those in the conventional method can be used. That is, as the buffer, for example, a buffer having a buffer action near neutral to alkaline, such as a phosphate buffer and a diethanol buffer, can be used, and its pH is 5.0 to 5.0.
11.0, more preferably about 7.0 to 10.0. At this time, the buffer concentration in the measurement solution is 10
It is not more than 00 mM, more preferably about 50 to 500 mM.

Examples of the tetrazolium dye include p-iodonitrotetrazolium violet (hereinafter abbreviated as INT), nitroble-tetrazolium, 2,6-dichlorophenolindophenol, and the like. Is about 0.1 to 10 mM, more preferably about 0.5 to 2 mM. The diaphorase is not particularly limited in its origin, but it is preferably a diaphorase derived from Bacillus stearothermophilus which has high stability, and its concentration in the measurement solution is 0.01 to 1000.
Unit / ml, more preferably 0.1-100 unit / ml
Degree. In this case, one unit of the diaphorase activity is defined as 1 μm of INT per minute at pH 8.0 and 30 ° C.
mol The amount of enzyme to be reduced .

As the surfactant, a nonionic surfactant or an ionic surfactant can be used. Among them, a Triton-based surfactant is preferable, and Triton X-100 is more preferable. The concentration in the measurement solution is 0.001 w / v% or more, and more preferably, about 0.02 to 1 w / v%. In the measurement method of the present invention, at the start of measurement of absorbance, NA is contained in the measurement solution.
It is sufficient that D or NADH and each of the above components are present, and there is no particular limitation on the order of mixing them.

The measuring method of the present invention can be specifically performed, for example, as follows. That is, at a temperature at which the enzyme activity is expressed at about 20 to 40 ° C., NAD or NA
400 μl of a buffer containing the above concentrations of ADH, a tetrazolium dye, diaphorase and a surfactant is added to 200 μl of the specimen containing DH, and colorimetric determination is performed. Examples of the colorimetric method include a rate assay method in which the change in absorbance is measured over time, and an end point assay method in which enzymatic cycling is performed for a certain period of time, the reaction is stopped by adding an acid, and the absorbance is measured. No. In either method, absorbance can be measured using a commercially available spectrophotometer.

Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.

[0016]

EXAMPLES Reference Example 1 Zymomonas mobilis ATCC 29191 was cultured in the following medium at 30 ° C. and pH 5.0. Medium composition 15% Glucose 0.5% Yeast extract 0.05% Potassium dihydrogen phosphate 0.05% Magnesium sulfate heptahydrate 0.001% Calcium pantothenate 0.00002% Bition After completion of culture, cells were collected by centrifugation. This cell 7
0.5 mM ammonium (II) sulfate, 10 mM in 0 g
Ascorbic acid, 0.1% Nonidet P-400.00
30 containing 02% DNaseI, 0.02% lysozyme
400 ml of mM potassium dihydrogen phosphate solution was added,
The suspension was incubated at 30 ° C. for 2 hours. The supernatant was obtained from this suspension by centrifugation. This supernatant is used for 30
mM sodium chloride, 2 mM magnesium chloride, 0.5
The pH was adjusted to 6.5 with Mes containing ammonium mM ferrous sulfate and 10 mM ascorbic acid. This was applied to a Blue Sepharose CL-6B (Pharmacia) column (diameter 3.0 cm × 15 cm), washed with 400 ml of the same buffer, eluted with 1 mM nicotine adenine dinucleotide (oxidized form), and its ADH activity was measured. The fractions were collected, concentrated and dialyzed to prepare ADH. Example 1 (1) Preparation of measurement solution A measurement solution having the following composition was prepared.

Sodium phosphate buffer (pH 8.0) 250 mM INT 1 mM ethanol 4% Triton X-100 0.1% Diaphorase (derived from Bacillus stearothermophilus, obtained from Seikagaku Corporation) 3 units / ml ADH ( 1.6 units / ml As a comparative example 1, a measurement solution having the same composition as described above except that ADH was derived from baker's yeast (obtained from Zicuma) was used. Prepared.

(2) Measuring method At room temperature, 100 μl of a measuring solution was added to a 96-well microtiter plate (manufactured by Corning), 10 μl of 1 μM NAD was added thereto, and then a microtiter plate automatic analyzer Biomec-1000 ( Beckman Co., Ltd.) for 0, 5, 15, 25 minutes and 49
The increase in absorbance per minute at 0 nm (ΔA ₄₉₀ ) was measured. From these measured absorbance values, the cycling rate (%) was calculated, with the time at 0 minute being 100.

(3) Results The measurement results are as shown in Table 1 and FIG.
As is clear from Table 1 and FIG. 1, in Comparative Example 1 using baker's yeast-derived ADH, the absorbance and the cycling rate rapidly decreased with time,
In the method of the present invention using ADH derived from Zymomonas mobilis (Example 1), the absorbance and the cycling rate were almost constant irrespective of the time. It was demonstrated that the detection sensitivity was more than 8 times and more than 8 times at 25 minutes.

[0020]

[Table 1]

Reference Example 2 Alkaline phosphatase (derived from calf small intestine, manufactured by Boehringer Mannheim) was added to N-succinimidyl 4- (N
-Maleimidomethyl) cyclohexane-1-carboxylate (3D Chemical Co., Ltd.) (3 mg) at pH 7.0,
The mixture was reacted at 30 ° C. for 10 minutes to introduce a maleimide group into alkaline phosphatase. Then, an anti-carcinoembryonic antigen antibody (hereinafter referred to as anti-CEA antibody) Fab ′ (prepared by digesting the antibody with pepsin and then reducing ) 1
mg was mixed at pH 6, and the maleimide group and the thiol group were crosslinked to prepare an alkaline phosphatase-labeled anti-CEA antibody. Example 2 Alkaline phosphatase was detected using the measurement method of the present invention.

50 μl of the alkaline phosphatase-labeled anti-CEA antibody prepared in Reference Example 2 was added to 0, 1.25,.
At a concentration of 5, 5.0, 10.0, 20.0 ng / ml,
The solution was added to each well of an anti-CEA antibody-immobilized microtiter plate (manufactured by Medix) and allowed to stand at room temperature for 2 hours to perform an antigen-antibody reaction. Then, a 20 mM Tris-HCl buffer (pH 7.0) containing 0.2% Tween 20, 0.2% bovine serum albumin, and 0.15 M sodium chloride.
Wash the plate with 2), and add 0.5 mM diethanolamine (pH
9.8) Add 50 μl to each well and add exactly 10 at room temperature.
After reacting for 1 minute, the same measurement solution as in Example 1 was added for 100 minutes.
Add 1 μl, react at room temperature for 10 minutes, and add 1N hydrochloric acid 50μ
The reaction was stopped by adding 1 l, and the respective absorbances (△
A ₄₉₀ ) was measured using a microtiter plate automatic analyzer Biomec-1000 (manufactured by Beckman). In addition, as Comparative Example 2, each absorbance when the same measurement solution as in Comparative Example 1 was used was measured in the same procedure as above.

The results of these measurements are shown in Table 2 and FIG. As is evident from these results, in the phosphatase detection system, the method of the present invention (Example 2) can obtain about four times the signal as compared with the conventional method (Comparative Example 2), and has excellent detection sensitivity. It was revealed.

[0024]

[Table 2]

[0025]

As described above in detail, in the assay method of the present invention, ADH which promotes enzymatic cycling
Since the activity does not decrease with time and the signal amplification reaction continues for a long period of time, NAD or NADH detection sensitivity equivalent to the conventional method can be obtained with a small amount of ADH. Also, AD
When the amount of H is the same, detection sensitivity far superior to the conventional method can be obtained.

[Brief description of the drawings]

FIG. 1 is a correlation diagram showing the change over time in the cycling rate in each of the method (●) of the present invention and the conventional method (○).

FIG. 2 is a correlation diagram showing the absorbance (A ₄₉₀ ) for each CEA concentration in each of the method (●) of the present invention and the conventional method (）).

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Claims (1)

(57) [Claims] 1. An enzymatic assay in a signal amplification system using nicotinamide adenine dinucleotide or its reduced form enzymatic cycling, wherein an alcohol dehydrogenase derived from Zymomonas sp. Is used as a catalyst for enzymatic cycling. Law.