JPH06141893A - Enzymic measuring method - Google Patents

Abstract

PURPOSE:To highly sensitively and stably detect NAD or NADH by specifying the catalyst of the enzymic cycling of the NAD or NADH to amplify the signal. CONSTITUTION:The enzymic cycling of NAD or NADH is performed in the presence of a thermophilic microorganism-originated amino acid dehydrogenase as a catalyst to amplify the color-generating signal. The enzymic activity of a measuring specimen is held during its storage, and the storage stability of the specimen is therefore excellent.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art NAD and its reduced form NADH
Is a coenzyme with the highest biological content, and is involved in the redox reaction of many dehydrogenases by reversibly changing through the exchange of hydrogen atoms. These NAD and N
Since ADH has absorption maxima near 260 nm and 340 nm, respectively, its absorbance change has been conventionally applied to the measurement of various enzyme activities. In addition to this, NAD or NADH can be detected with high sensitivity. As a measuring 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, Volume 148, Volumes 119-12
Page 4, 1985: FJ Dhahir et al., Clinical Chemistr.
y, 38 (2), pages 227-232, 1992.
Year).

In this assay method, a signal that develops in color along with the oxidative ring reaction of NAD or NADH is amplified by repeating the reaction (enzymatic cycling) to obtain NA.
The presence of D or NADH is detected 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 at that time, NAD is reduced. Furthermore, this NADH oxidizes to NAD again in the presence of a tetrazolium dye and its dibasic enzyme diaphorase.
At the same time, the tetrazolium-based dye is cyclized to generate formazan and a colored signal is obtained. Since NAD repeats redox under such conditions, the signal intensity gradually increases. When NADH is present in the sample, it develops color by the same principle and amplifies its intensity.

[0004]

[Chemical 1]

NAD or NADH by such a system
Is detected by measuring the activity of phosphatase (CHSelf et al., Supra: FJDhahir et al., Supra) and the activity of NAD synthetase (Hideo Misaki, BIO INDUSTRY, Volume 7,
Pp. 775-787, 1990) and the like, and particularly in the measurement of alkaline phosphatase, the detection sensitivity equivalent to that of the fluorescence method or the coloring method can be obtained despite the colorimetric method. It is positioned as a very excellent method in the field (Ishikawa et al., "Immunoassay 3rd Edition", pages 58-60, published by Ikusho Shoin).

Conventionally, baker's yeast-derived one has been used as ADH in such a measurement system using enzymatic cycling. However, since the activity time of this baker's yeast-derived ADH is short, a large amount of ADH must be added to the measurement reagent in order to repeatedly oxidize NAD or NADH to obtain a sufficient chromogenic signal. It was

On the other hand, the inventors of the present invention
When Zymomonas-derived ADH is used as a catalyst for the enzymatic cycling of AD or NADH,
It was found that an excellent chromogenic signal can be obtained by adding a small amount of ADH as compared to conventional baker's yeast-derived ADH, and based on this finding, a highly sensitive enzymatic assay method was invented and a patent application has already been filed (special Japanese Patent Application No. 4-60743).

As a catalyst for the enzymatic cycling of NAD or NADH, ADH is generally used because of its high specificity.

[0009]

[Problems to be Solved by the Invention] However, ADH
However, when added to a measuring reagent for NAD or NADH, the enzyme activity of the measuring reagent decreases with time, and thus the measuring reagent has a short effective period. This invention
It was made in view of the above circumstances.
It is an object of the present invention to solve the drawbacks of the conventional enzymatic assay method using EDTA and to provide a new enzymatic assay method capable of stably detecting NAD or NADH with high sensitivity.

[0010]

As a solution to the above problems, the present invention is suitable as a catalyst for enzymatic cycling in a signal amplification system using enzymatic cycling of nicotinamide adenine dinucleotide or its reduced form. Provided is an enzymatic measuring method characterized by using an amino acid dehydrogenase derived from a thermophilic microorganism.

The structure and preferred embodiments of the present invention will be described in more detail below. Detection of NAD and NADH in the enzymatic assay method of the present invention is carried out by using a sample containing any one of these, a thermophilic microorganism-derived amino acid dehydrogenase and its substrate (corresponding amino acid), diaphorase, a tetrazolium dye, an interface. The absorbance change of formazan caused by the ring of the tetrazolium-based dye may be measured by mixing with a measurement reagent such as an activator and a buffer.

In the present invention, the thermophilic microorganism-derived amino acid dehydrogenase used as a measuring reagent component is, for example, alanine dehydrogenase (hereinafter abbreviated as AlaDH),
Leucine dehydrogenase (hereinafter abbreviated as LeuDH),
Glutamate dehydrogenase and the like can be used by culturing thermophilic microorganisms such as Bacillus stearothermophilus by a conventional method and extracting and purifying from the culture solution. The concentration of these amino acid dehydrogenases in the measuring reagent is 0.01 to 1000 units / ml, more preferably 0.1 to 100 units / ml. As the substrate for these amino acid dehydrogenases, amino acids depending on the type of dehydrogenase used can be used, and the concentration thereof is 1 to 1000 mM, more preferably 10 to 100 mM.
Set to mM. One unit of activity of amino acid dehydrogenase is defined as the amount of enzyme that oxidizes 1 μmol of the corresponding amino acid per minute at pH 9.0 and 30 ° C.

As the components other than the amino acid dehydrogenase and the amino acid in the measuring reagent, the same ones as in the conventional method can be used. That is, as the buffer solution, for example, a buffer solution having a buffer function in the vicinity of neutrality, such as a phosphate buffer solution or a triethanol buffer solution, can be used.
H is 5.0 to 10.0, and more preferably 7.0 to 9.0.
The degree. The concentration of the buffer solution in the measuring reagent at this time is 10 to 1000 mM, more preferably 50 to 500 mM.
It is about mM.

As the tetrazolium dye, 2- (P
Nitrophenyl) -3- (P-iodophenyl) -5-
Phenyltetrazolium chloride (hereinafter abbreviated as INF), 3,3 ′-(3,3′-dimethoxy-4,
4'-diphenylene) bis (2- (P-nitrophenyl) -5-phenyl-tetrazolium chloride), 2
-(4 ', 5'-dimethyl-2'-thiazolyl-3,
5, -diphenyltetrazolium bromide and the like can be exemplified, and the concentration in the measuring reagent is 0.1 to 10 m.
M, more preferably about 0.5 to 2 mM.

The diaphorase is not particularly limited in its origin, but is preferably highly stable, for example, derived from Bacillus stearothermophilus, and its concentration in the measuring reagent is 0.01 to 1000 units / ml. It is more preferably about 0.1 to 100 units / ml. In this case, 1 unit of diaphorase activity means pH 8.0, 30 ° C.
It is the amount of enzyme that 1 μmol of INT is recovered per 1 minute.

As the surface active agent, a nonionic surface active agent or an ionic surface active agent can be used. Of these, a Triton type surface active agent is preferable, and a Triton X-100 is more preferable. The concentration in the measuring reagent is 0.001 w / v% or more, more preferably about 0.02 to 1 w / v%. In the measuring method of the present invention, NA is added to the measurement solution at the start of measuring the absorbance.
It suffices that D or NADH and the above components are present, and there is no particular limitation on the mixing order of them.

The measuring method of the present invention can be specifically carried out as follows, for example. That is, at a temperature of about 20 to 40 ° C at which enzyme activity is expressed, NAD or NA
To 200 μl of a sample containing DH, 400 μl of a buffer solution containing the above-mentioned concentrations of amino acid dehydrogenase, amino acid, tetrazolium dye, diaphorase and a surfactant is added, and colorimetric determination is performed. Examples of the colorimetric method include a rate assay method in which a change in absorbance is measured with time, or an end point assay method in which the reaction is stopped by adding an acid after enzymatic cycling for a certain period of time to measure the absorbance. Can be mentioned. In either method, the 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.

[0019]

Examples Example 1 (1) Preparation of measurement reagent A measurement reagent having the following composition was prepared. Sodium phosphate buffer (pH 8.0) 250 mM INT 1 mM alanine 50 mM Triton X-100 0.1% diaphorase (from Bacillus stearothermophilus) 1 unit / ml Bacillus stearothermophilus derived AlaDH 1 unit / ml As a comparative example, baker's yeast-derived ADH was used for dehydrogenase.
And a measurement reagent having the same composition as the above except that ethanol was used as the substrate.

(2) Measuring method Reagents prepared in the above (1) (Example 1 and Comparative Example)
Were incubated at 37 ° C. and sampled over time to measure the enzyme activity and DIA of each. (3) Results The measurement results are as shown in Table 1 and FIG.
As is clear from Table 1 and FIG. 1, AlaD
The decrease in enzyme activity over time during storage of the reagent containing H (Example 1) was about 1/10 or less of that of the reagent containing ADH derived from baker's yeast (Comparative Example). From the above results, it was confirmed that AlaDH, when used as a measurement reagent component, maintains the enzyme activity for a much longer period than ADH.

When the measurement was performed using other thermophilic microorganism-derived amino acid dehydrogenase, almost the same results were obtained.

[0022]

[Table 1]

Reference Example 2 mg of alkaline phosphatase (derived from calf small intestine, manufactured by Boehringer Mannheim) and 3 mg of succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (manufactured by G-Chem Chemical) at pH 7.0 and 30.
The reaction was performed at 10 ° C for 10 minutes to introduce a maleimide group into alkaline phosphatase. Then, 1 mg of anti-carcinoembryonic antigen antibody (hereinafter referred to as anti-CEA antibody) Fab ′ (prepared by digesting antibody with pepsin and then reducing)
Was mixed at pH 6 to crosslink the maleimide group and the thiol group to prepare an alkaline phosphatase-labeled anti-CEA antibody. Example 2 Carcinoembryonic antigen (hereinafter abbreviated as CEA) 50 μl
0, 0.25, 0.5, 1.0, 2.5, 5.0, 1
Anti-CEA antibody-immobilized microtiter plate (Medix) at each concentration of 0.0 and 20.0 ng / ml.
Alkaline phosphatase-labeled anti-CEA antibody 5 prepared in the reference example.
0 μl was added at a concentration of 1 μg / ml, and the mixture was allowed to stand at room temperature for 2 hours for antigen-antibody reaction. Then, a 20 mM Tris-HCl buffer solution (pH: 0.2% Tween 20, 0.2% bovine serum albumin, 0.15 M sodium chloride)
The plate was washed with 7.2) and 0.5 μM diethanolamine (pH 9.8) containing 1 mM nicotinamide adenine dinucleodolinic acid and 1 mM magnesium chloride (50 μm).
1 was added to each well and reacted at room temperature for exactly 10 minutes. Then, to this reaction solution, 100 μl of the same measurement reagent as in Example 1 was added immediately after the adjustment or after the adjustment and after incubation at 37 ° C. for 288 hours, the reaction was performed at room temperature for 10 minutes, and 50 μl of 1N hydrochloric acid was added to stop the reaction. Then, the respective absorbances (ΔA ₄₉₀ ) were measured using a microtiter plate automatic analyzer Biomec-1000 (manufactured by Beckman).
Was measured using. As a control, each absorbance when the measurement reagent of the comparative example of Example 1 was used was measured by the same procedure as above.

The results of these measurements are as shown in Table 2 and FIG. As is clear from these results, C
In the EA detection system, the method of the present invention (Example 2) and the conventional method (Comparative Example) have almost the same detection sensitivity when the respective measurement reagents are used immediately after preparation (0 hours). There is. However, when the measurement reagent is stored for 288 hours, the activity of the reagent of the conventional method almost disappears, whereas the reagent used in the present invention still maintains the residual activity of about 80%, Its excellent stability was demonstrated.

Similar results were obtained when other amino acid dehydrogenases derived from thermophilic microorganisms were measured.

[0026]

[Table 2]

Example 3 AlaDH derived from Bacillus stearothermophilus was
A measurement reagent having the same composition as in Example 1 was prepared, except that LeuDH of the same origin was used. As a control, the same reagent as that of the comparative example of Example 1 was prepared. These measurement reagents were incubated at 37 ° C. for 96 hours, and then used in Example 2
The same CEA detection system was used.

The results are as shown in Table 3 and FIG. As is clear from these results, it is apparent that the method of the present invention can obtain a signal about 10 times higher than that of the conventional method even after storing the measuring reagent for 96 hours, and maintain excellent detection sensitivity. became.

[0029]

[Table 3]

[0030]

INDUSTRIAL APPLICABILITY As described in detail above, in the assay method of the present invention, the enzyme activity of the assay reagent used is maintained even during storage, so that a signal amplification system excellent in storage stability is established. As a result, the measurement reagent, which has conventionally required adjustment at the time of use, can be stored at room temperature for at least about 2 weeks, and the troublesome preparation of the reagent can be omitted.

[Brief description of drawings]

FIG. 1 is a correlation diagram showing changes over time in enzyme activity in each of the method (●) of the present invention and the conventional method (◯).

FIG. 2 shows the case where the measurement reagent used in the method of the present invention is stored for 0 hours (▲) and 288 hours (●), and the case where the reagent of the conventional method is 0 hours (Δ) and 288 hours (◯).
FIG. 3 is a correlation diagram showing the absorbance (A ₄₉₀ ) for each CEA concentration in each case of storage.

FIG. 3 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 (◯).

Claims (1)

[Claims] 1. A signal amplification system using enzymatic cycling of nicotinamide adenine dinucleotide or its ring form, wherein an amino acid dehydrogenase derived from a thermophilic microorganism is used as a catalyst for enzymatic cycling. Measurement method.