JPH08248028A - Liquid reagent for measuring physiologically active substances - Google Patents

Abstract

(57) [Summary] [Object] To provide a liquid reagent capable of accurately measuring a physiologically active substance even when stored in a solution state for a long time. A liquid reagent for measuring a physiologically active substance containing NADH or NADPH contains a dehydrogenase having NAD or NADP as a coenzyme and a substrate for this dehydrogenase. [Effect] The amount of NADH or NADPH can be constantly maintained for a long period of time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a liquid reagent for measuring a physiologically active substance containing reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate. More particularly, it relates to an improved liquid reagent for measuring a physiologically active substance in which reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate is stable for a long period of time in a solution state.

[0002]

2. Description of the Related Art Many clinical test agents used in hospital laboratories and test centers use reagents such as unstable enzymes and substrates. Generally, in consideration of their stabilization. It is supplied as a lyophilized product, and it is predominantly used after being dissolved in purified water or a specific buffer solution at the time of use. However, when many items are measured and the number of samples increases, the work of dissolving the freeze-dried product often becomes a burden.
In addition, the stability of the reagent after dissolving the freeze-dried product is insufficient, and it is desired to develop a liquid reagent that is stable in a solution state for a long period of time without the need to prepare the reagent at the time of use.

In particular, aspartate aminotransferase (GOT), alanine aminotransferase (GPT), lactate dehydrogenase (LD) in a biological sample solution.
H), α-oxybutyrate dehydrogenase (HBD), free fatty acid (NEFA), urea nitrogen (UN), creatinine (CRE) and other physiologically active substances are routinely used as important indicators for diagnosis of various diseases. It is the item being measured. The reagent for measuring the physiologically active substance in the biological sample liquid is a reduced nicotinamide adenine dinucleotide (hereinafter, abbreviated as NADH) or a reduced nicotinamide adenine dinucleotide phosphate (hereinafter, N) as a common component.
(Abbreviated as ADPH). Therefore, when preparing a liquid reagent for measuring the various physiologically active substances described above,
These NADH or NADPH [hereinafter, NAD (P) H
Is abbreviated as "].

Generally, NAD (P) H can be maintained to some extent in stability if they are stored in an alkaline solution. Further, as a method for stabilizing NAD (P) H, a method in which a specific base (amine base such as imidazole or strong base such as sodium hydroxide) is allowed to coexist (JP-A-55-42599).
JP), a method of adding a chelating agent such as EDTA and an azide such as sodium azide (JP-A-59-8239).
No. 8), a method of adding boric acid (JP-A-62-19).
8697), and a method of coexisting an alkali metal or ammonium bicarbonate buffer (Japanese Patent Laid-Open No. 4-229).
No. 192) has been reported.

[0005]

However, as a reagent for measuring a physiologically active substance in a biological sample liquid, NAD is used.
In addition to (P) H, other substances such as enzymes and substrate components necessary for expressing a desired reaction are also included.
Of course, it is necessary to consider the maintenance of the reaction atmosphere (pH range, etc.) and the stability of other components.
It is not the case that an optimal stabilization method should be taken against. The above-described conventional stabilization method has insufficient long-term stability of NAD (P) H, particularly in a solution state.

[0006] For example, although NAD (P) H is considerably stable in an alkaline solution, coexisting contaminants (especially dehydrogenase having NAD (P) H as a coenzyme), buffer concentration, and pH over time. Due to various factors such as fluctuations,
Although gradually, oxidized nicotinamide adenine dinucleotide (hereinafter, abbreviated as NAD) or reduced nicotinamide adenine dinucleotide phosphate (hereinafter, NA)
(Abbreviated as DP). NAD to save
In consideration of the rate of decrease of (P) H, the amount of NAD (P) H added is increased in advance so that the amount of NAD (P) H necessary for a desired reaction can be secured during actual measurement. However, such a method is not preferable in terms of cost. Furthermore, it is also inconvenient for a reagent that measures the change in absorbance of NAD (P) H at 340 nm.

Therefore, an object of the present invention is to stabilize NAD (P) H contained in a reagent for measuring a physiologically active substance so that it can be used as an index for diagnosing a disease or the like even if it is stored for a long time in a solution. Another object of the present invention is to provide a liquid reagent for measuring a physiologically active substance, which can accurately measure the following physiologically active substance.

[0008]

The above object is to provide a liquid reagent for measuring a physiologically active substance containing at least reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate according to the present invention, wherein oxidized nicotine is used. This can be achieved by a liquid reagent characterized by containing a dehydrogenase having amide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate as a coenzyme and a substrate for this dehydrogenase.

The present invention will be described in detail below. The combination of dehydrogenase having NAD or NADP [hereinafter abbreviated as NAD (P)] as a coenzyme and its substrate is not particularly limited, but the dehydrogenase and its substrate have a pH of 8.5.
It is preferable that it is stable between 11 and 11, and it can be selected according to the measurement system. As such a combination,
For example, glucose-6-phosphate dehydrogenase (hereinafter, G
6PDH) and glucose-6-phosphate (hereinafter abbreviated as G6P) or a salt thereof, glutamate dehydrogenase (hereinafter abbreviated as GlDH) and L-glutamic acid or a salt thereof, or isocitrate dehydrogenase. (Hereinafter, abbreviated as ICDH) and isocitric acid or its salt can be used. The dehydrogenase and substrate using NAD (P) as a coenzyme are trace amounts of NAD (P) H
Can be suppressed. The specific amount to be added can be appropriately determined according to the individual measurement system used.

For example, when G6PDH and G6P or a salt thereof (eg, potassium salt, sodium salt, barium salt) is used as a dehydrogenase having NAD (P) as a coenzyme and a substrate thereof, the G6PDH activity is 0.001 U. /
When it is 1 or more, it has an effect of suppressing the decrease of NAD (P) H, and 0.01 U / l to 1.0 U / l is particularly preferable. G6P or a salt thereof is effective at 0.05 mM or more, and particularly, it is less than 0.
1 mM to 10 mM is preferable. G6PDH activity is 1.0
When it exceeds U / l or when the concentration of G6P exceeds 10 mM, ammonia is produced when urease comes into contact with urea in the sample, and NA against the production rate of ammonia is increased.
The rate of decrease of D (P) H may be delayed. In addition, G
As 6PDH, G6P or its salt is used as a substrate and NA
It is not particularly limited as long as it uses D (P) as a coenzyme. For example, yeast and Leuconostoc mesenteroides (Leuconostoc mesenteroides) can be used.
es) -derived G6PDH can be preferably used.

[0011] For example, when GlDH and L-glutamic acid or a salt thereof (eg, potassium salt, sodium salt) are used as a dehydrogenase having NAD (P) as a coenzyme and its substrate, the GlDH activity is 0. 005 U / l
As described above, there is an effect of suppressing the decrease of NAD (P) H, and 0.05 U / l to 50 U / l is particularly preferable. L-glutamic acid or its salt is effective at 0.1 mM or more, and particularly preferably 5 mM to 50 mM. GLDH activity is 50U
If it exceeds / l, the composition components may deteriorate due to contamination with protease and other dehydrogenases, and if L-glutamic acid or its salt exceeds 50 mM, the solubility becomes poor. In addition, as GlDH, NAD
The origin is not particularly limited as long as it uses (P) H as a coenzyme and α-ketoglutarate and ammonia as substrates. Specifically, for example, yeast or Proteus (Pro
teus) -derived GlDH derived from a microorganism can be preferably used.

Further, for example, a dehydrogenase having NAD (P) as a coenzyme and ICDH as its substrate and isocitrate or its salt (for example, sodium salt, potassium salt)
When using, it is preferable to add a metal ion such as a magnesium ion or a manganese ion. In this case, ICDH activity was 0.001 U / l or more and NAD
It has the effect of suppressing the decrease of (P) H, especially 0.1 U / l
-100 U / l is preferred. Isocitric acid or its salt is effective at 0.01 mM or more, especially 0.1 mM
-10 mM is preferred. When magnesium chloride is used as the metal ion, an effect is observed at 0.1 mM or more, and 0.5 mM to 50 mM is particularly preferable. The effect of isocitrate or its salt is observed at 0.05 mM or more, and particularly, it is 0.1.
5 mM to 50 mM is preferable. ICDH activity is 50 U /
When it exceeds 1, the composition components may deteriorate due to contamination with protease and other dehydrogenases. When magnesium chloride exceeds 50 mM, the solubility becomes poor, and when isocitrate or its salt exceeds 50 mM. Solubility deteriorates.

The liquid reagent for measurement of the present invention is NAD (P)
In order to maintain the stability of H, preferably pH 7 to 1
2, more preferably within the range of 8.5 to 11, depending on the measurement system. In the present specification, the “physiologically active substance” is not particularly limited, but clinically present in a biological sample (eg, blood, serum, plasma, urine, spinal fluid, cell or tissue extract, saliva, sweat). It means any substance to be tested, such as various enzymes. For example, aspartate aminotransferase (GOT), alanine aminotransferase (GPT), lactate dehydrogenase (LDH), α-oxybutyrate dehydrogenase (HB
D), free fatty acid (NEFA), urea nitrogen (UN), creatinine (CRE) and the like.

In the above-mentioned conventional method, the main purpose is to keep the decomposition of NAD (P) H to a minimum by adjusting the liquidity and additives. On the other hand,
The present invention allows NAD (P) H generated from NAD (P) H to coexist with a dehydrogenase having NAD (P) as a coenzyme and a substrate of this enzyme, forcibly converting it into NAD (P) H. By returning, the amount of NAD (P) H is constantly maintained over a long period of time. This is achieved only because the reagent for measuring a physiologically active substance in a biological sample solution, which is the object of the present invention, is in a solution state, and such an idea per se is not seen in the conventional method. .

The liquid reagent for measuring a physiologically active substance of the present invention is
In a liquid reagent for measuring a physiologically active substance in a biological fluid, it becomes possible to constantly maintain the amount of NAD (P) H for a long period of time. Particularly, specific examples of the measurement system to which the present invention can be preferably applied are shown below.

(1) Reagent for measuring aspartate aminotransferase (GOT) The essential constituents of this measuring reagent are L-aspartic acid, α-ketoglutarate, malate dehydrogenase and NA.
D (P) H, which are appropriately constituted in a two-reagent system, and a dehydrogenase having NAD (P) as a coenzyme and its substrate coexist in a reagent containing at least NAD (P) H.

(2) Lactate dehydrogenase (LDH) assay reagent The essential components of this measuring reagent are pyruvic acid and NA.
D (P) H, which is a one-reagent system or an appropriate two-reagent system, and which contains at least NAD (P) H
A dehydrogenase having AD (P) as a coenzyme and its substrate are allowed to coexist.

(3) Reagent for measuring alanine aminotransferase (GPT) The essential components of this measuring reagent are L-alanine and α-
Ketoglutaric acid, lactate dehydrogenase and NAD (P) H, which are appropriately constituted in a two-reagent system and have at least NA
A dehydrogenase having NAD (P) as a coenzyme and its substrate coexist in a reagent containing D (P) H.

(4) α-oxybutyrate dehydrogenase (HB
D) Reagent for measurement The essential constituents of this measuring reagent are α-ketobutyric acid and N
AD (P) H, which is a one-reagent system or an appropriate two-reagent system, and which contains at least NAD (P) H,
A dehydrogenase having NAD (P) as a coenzyme and its substrate coexist.

(5) Reagent for measuring free fatty acid (NEFA) The essential constituents of this measuring reagent are ATP, CoA (Coenzyme A), acyl-CoA synthetase, myokinase, phosphoenolpyruvate, pyruvate kinase, LDH and NAD (P) H, and these are appropriately used.
A dehydrogenase having NAD (P) as a coenzyme and its substrate coexist in a reagent containing a reagent system containing at least NAD (P) H.

(6) Urea nitrogen (UN) measuring reagent The essential components of this measuring reagent are glutamate dehydrogenase (GlDH), α-ketoglutarate, urease and NAD (P) H, and these are appropriately constituted in a two-reagent system, and at least NAD (P) H Including reagents, NAD
A dehydrogenase having (P) as a coenzyme and its substrate coexist.

(7) Creatinine (CRE) measuring reagent-1 The essential components of this measuring reagent are creatinine amidohydrolase, creatine kinase, ATP, pyruvate kinase, phosphoenolpyruvate, LDH and N.
AD (P) H, which are appropriately configured in a two-reagent system,
To a reagent containing at least NAD (P) H, NAD (P)
Coexist with a dehydrogenase and its substrate.

(8) Creatinine (CRE) measuring reagent-2 The essential components of this measuring reagent are creatinine deiminase, α-ketoglutarate, urease and NAD.
(P) H, which are appropriately constituted in a two-reagent system, and a dehydrogenase having NAD (P) as a coenzyme and its substrate coexist in a reagent containing at least NAD (P) H.

[0024]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1: Measurement of residual rate of NADPH Liquid reagent having composition shown in Table 1 (hereinafter referred to as control reagent 1), liquid reagent having composition obtained by removing α-ketoglutaric acid and GlDH from the composition shown in Table 1 ( Below, control reagent 2
0.01 U / l-G6 in the composition shown in Table 1.
The liquid reagent of the present invention having a composition to which PDH and 0.1 mM-G6P are added (hereinafter, referred to as Reagent 1 of the present invention), and the liquid reagent of the present invention having a composition to which 10 mM-L-glutamic acid is added to the composition shown in Table 1. (Hereinafter, referred to as Reagent 2 of the present invention), Table 1
A liquid reagent of the present invention (hereinafter referred to as the present reagent 3) having a composition obtained by adding 10 U / l-ICDH, 1.0 mM isocitrate, and 1.0 mM magnesium chloride to the composition shown in (3) was prepared. Immediately after preparation, each liquid reagent was further stored in a solution state at 10 ° C., and after one month, two months, four months, and six months, the absorbance at 340 nm was measured to determine the remaining amount of NADPH. Absorbance on the day of preparation is 100% and 1
Table 2 shows the survival rates after months, 2 months, 4 months and 6 months.

[0025]

[Table 1] Triethanolamine hydrochloride buffer (pH 9.0) 50 mM NADPH 0.3 mM α-ketoglutarate 5.0 mM GlDH 20 U / l

[0026]

[Table 2] Storage period (month) 0 1 2 4 6 Control reagent 1 100 96 96 92 85 85 78 Control reagent 2 100 94 94 88 82 82 73 Inventive reagent 1 100 98 98 96 90 90 85 Inventive reagent 2 100 96 96 94 86 82 Inventive Reagent 3 100 97 94 88 84

Example 2: Residual rate of measured UN amount A solution having the composition shown in Table 3 (hereinafter referred to as the second reagent) was prepared. This second reagent and each liquid reagent prepared in Example 1 were stored at 10 ° C., before storage (immediately after preparation) and after 1 month,
The respective reagents after 2 months, 4 months and 6 months were used for the UN amount measurement shown below. Urea nitrogen content is 150mg / d
A urea aqueous solution of 1 was prepared and used as a test sample.
Test sample 4 was added to 320 μl of each liquid reagent prepared in Example 1.
μl was added, mixed by stirring, and heated at 37 ° C. for 5 minutes,
80 μl of the second reagent was added, mixed by stirring, and heated at 37 ° C. for 5 minutes. The amount of change in absorbance per minute at 340 nm from 1 minute to 3 minutes after the addition of the second reagent was measured. Absorbance change immediately after preparation is 100%,
Table 4 shows the residual rates of the amount of change in absorbance after one month, two months, four months, and six months.

[0028]

[Table 3] Triethanolamine hydrochloric acid buffer solution (pH 7.80) 100 mM urease 1.3 U / ml acetohydroxamic acid 0.4 mM

[0029]

[Table 4] Storage period (month) 0 1 2 4 6 Control reagent 1 100 95 95 91 78 78 44 Reagent 1 of the present invention 1 100 97 97 95 94 91 91 Reagent 2 of the present invention 2 100 94 94 93 86 77 Reagent 3 100 96 96 94 92 89

[0030]

INDUSTRIAL APPLICABILITY The liquid reagent for measuring a physiologically active substance of the present invention is a liquid reagent for measuring a physiologically active substance in a biological fluid, which constantly maintains the amount of NAD (P) H for a long period of time. Is possible.

Claims (4)

[Claims] 1. A liquid reagent for measuring a physiologically active substance, which comprises at least reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate, comprising: oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphorus A liquid reagent comprising a dehydrogenase having an acid as a coenzyme and a substrate for the dehydrogenase. 2. The liquid reagent according to claim 1, wherein the dehydrogenase is glucose-6-phosphate dehydrogenase and the substrate thereof is glucose-6-phosphate or a salt thereof. 3. The dehydrogenase is glutamate dehydratase, and the substrate thereof is glutamic acid or a salt thereof.
The liquid reagent according to. 4. The liquid reagent according to claim 1, wherein the dehydrogenase is isocitrate dehydrogenase, the substrate thereof is isocitrate or a salt thereof, and further contains a metal ion.