JP3442182B2 - Substrate reagent of two-reagent liquid reagent and method for stabilizing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for stabilizing 2-oxoglutarate and a stable liquid reagent containing 2-oxoglutarate obtained by the method.

[0002]

2. Description of the Related Art In the medical field, it is necessary to accurately diagnose a disease in order to carry out an appropriate treatment. Accurate, rapid, and accurate measurement of many index substances contained in biological samples
It is desired to measure easily, and various test methods, reagents and analytical instruments have been developed. In recent years, it has become mainstream to process a large number of specimens in a short time by an automatic inspection device and automatically obtain measurement results, and higher performance is required for both hardware (inspection device) and software (reagent). There is. In particular, regarding reagents, conventionally, in consideration of the storage stability of reagent compositions such as enzymes and substrate substances, in many cases, reagents have been supplied as freeze-dried products and used by dissolving them at the time of inspection. In order to save the labor at the time of inspection, it is desired that the reagent form be supplied in a liquid state from the beginning.

The substances to be measured are diverse.
Many indicator substances have been measured. Among them, glutamate pyruvate transaminase (hereinafter, also referred to as GPT) and glutamate oxaloacetate transaminase (hereinafter, also referred to as GOT) are enzymes that are widely distributed in the heart and liver, and are translocated into the blood during various diseases. The measurement of GPT and GOT in biological fluids such as blood and blood is frequently measured as one of the important items as an index for the follow-up observation of diagnosis and treatment of heart disease and liver disease.

As a method for measuring GPT, L-alanine and 2-oxoglutarate (α-ketoglutarate; hereinafter referred to as α)
-KG) is used as a substrate, pyruvate produced by GPT is converted to lactate by lactate dehydrogenase (hereinafter abbreviated as LDH), and coexisted reduced nicotinamide adenine dinucleotide (hereinafter, abbreviated as NAD
A method of measuring GPT by measuring the amount of decrease in the amount (abbreviated as H) in the vicinity of a wavelength of 340 nm is widely used.

The reaction equation is shown below. NAD is an oxidized nicotinamide adenine dinucleotide (the same applies hereinafter).

As a method for measuring GOT, oxaloacetate produced by GOT is treated with malate dehydrogenase (hereinafter, referred to as M) by using L-aspartic acid and α-KG as substrates.
A method for measuring GOT by measuring the amount of reduction of reduced nicotinamide adenine dinucleotide (NADH) coexisted with malic acid by abbreviated as DH) at a wavelength of about 340 nm is widely used. .

The reaction equation is shown below.

As mentioned above, reagents containing enzymes are generally
In consideration of its storage stability, it was provided as a lyophilized product containing the composition necessary for the reaction, and it was used by dissolving it in a buffer solution or the like at the time of use. However, from the viewpoint of workability and cost, it has become necessary to supply in the form of a liquid reagent in advance. Since these reagents are often used in automatic analyzers, it is necessary to maintain the stability of the reagent composition for a long period (for example, half a year to one year) by using a two-reagent system as the reagent composition. Particularly, in the solution state, the stability of the enzymes is remarkably lowered, so that the optimum pH of the solution is set, various stabilizers are added, and the like, and some results have been obtained.

On the other hand, the substrate substances (L-alanine or L-aspartic acid and α-KG in the case of the above-mentioned measurement examples of GPT and GOT) have been studied for stabilization as much as enzymes. Absent. This is because, in general, many of the substrate substances are chemically stable substances, do not decompose rapidly like enzymes, and are designed to be added in large excess as a reagent composition. Therefore, even if the decomposition proceeds to some extent, if a sufficient amount remains to express the desired reaction at the time of measurement,
Since it does not interfere with the detection of the target substance, compared to the enzyme,
It was not so important in terms of stabilization.

For example, JP-A-57-39799 and JP-A-57-39800 disclose GOT and GPT.
Techniques for reagent stability are described. Especially LD
In order to stabilize H, not only conventional bactericides but also sulfhydryl compounds (eg reduced glutathione, N-acetylcysteine, etc.) and chelating agents (eg EDTA, EGTA) are used in combination / addition of LDH. This is for stabilization. However, the technology described in these patent publications is a three-reagent system, and the storage stability in a solution state is only about 10 days at room temperature. Further, the focus is on the stability of enzymes such as LDH, and regarding the stability of other compounding components necessary for GOT and GPT measurement, for example, NADH, simply under alkaline conditions of pH 9 to 11, There is no problem in lifespan, especially for the substrate α-KG, it is possible to use a bactericide (alkali metal azide, cationic surfactant, benzoic acid derivative, methyleneglutaronitrile bromide, various antibiotics, etc.). It just states that there is no problem with its stability.

[0011]

However, according to the confirmation by the present inventors, for example, the substrate α-, which has not been so much emphasized in its stability in the past, was used.
When a liquid reagent to which an excess amount of KG was added was prepared and the content after storage for a long period of time was measured, it was found that the amount of α-KG decreased depending on the composition condition of the reagent. It was also found to affect. For example, the above-mentioned GOT and GPT measurement reagents are typical examples of diagnostic measurement reagents that use α-KG as a reagent component. As these measuring reagents, there are various commercially available products, and for example, there are those which are composed of a two-reagent system in which enzymes and substrates are separated as shown below.

Reagent for GOT measurement First reagent: NADH: 0.35 mM MDH: 1000 U LDH: 1000 U 20 mM glycine buffer (pH 9.2) Second reagent: α-KG: 31 mM L-aspartic acid: 400 mM 100 mM HEPES buffer ( pH 7.8) [HEPES = N-2-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid]

Reagent for measuring GPT First reagent: NADH: 0.35 mM LDH: 5000U 20 mM glycine buffer (pH 9.2) Second reagent: α-KG: 47 mM L-alanine: 1000 mM 100 mM HEPES buffer (pH 7.5)

The present inventor has made the above GOT measuring reagent and G
When the storage stability was traced for the α-KG substrate solution of each second reagent of the PT measurement reagents,
In the measurement reagent, the amount of α-KG immediately after preparation is 100%.
It was confirmed that in the case of the above, the temperature decreased by 20% or more in about 5 months (25 ° C). Regarding the GOT reagent, although it was not as great as that of the GPT reagent, it clearly showed a significant decrease tendency. For reagents stored for a longer period of time, α-
The amount of KG will decrease. When the residual amount of α-KG decreases to 50%, GOT and GPT activity values are measured as low values of about 3 to 5%, which is a great influence in cases where accurate diagnosis is required.

As mentioned above, conventionally, in particular, the substrate α-KG
In contrast, it is considered that there is no problem in its stability if a bactericide (alkali metal azide, cationic surfactant, benzoic acid derivative, methyleneglutaronitrile bromide, various antibiotics, etc.) is used. However, depending on the composition conditions of the reagent, when a liquid reagent in which α-KG and a substrate amino acid are combined is adopted when the reagent is composed of the two-reagent system as described above, the decrease of α-KG is noted. The need arises.

On the other hand, the present inventor has proposed that the reagent component α
As a result of a study aimed at stabilizing -KG, it was surprisingly found that, in the case of a structure in which amino acids coexist, α-KG can be kept stable for a long period of time by adding a chelating agent. Therefore, an object of the present invention is to establish a method for stabilizing α-KG, stabilize a liquid reagent containing α-KG, and provide a highly reliable reagent.

[0017]

The present invention is a method for stabilizing a substrate reagent in a two-reagent liquid reagent composed of an enzyme and a substrate, wherein the substrate reagent is an object to be measured. To contain 2-oxoglutarate (α-KG) and L-alanine or L-aspartate as a substrate for the enzyme glutamate pyruvate transaminase (GPT) or glutamate oxaloacetate transaminase (GOT), and to coexist with a chelating agent. The present invention relates to a method for stabilizing a substrate reagent as described above. Furthermore, the present invention is a substrate reagent in a two-reagent liquid reagent composed of an enzyme and a substrate, wherein the substrate reagent is glutamic acid pyruvate transaminase (GPT) which is an enzyme to be measured. Glutamate oxaloacetate transaminase (GOT) contains 2-oxoglutarate (α-KG) as a substrate and L-alanine or L-aspartic acid, and coexists with a chelating agent. It also concerns.

The present invention will be described in detail below. The chelating agent of the present invention is a GOT containing α-KG and an amino acid.
Alternatively, it is allowed to coexist with a liquid reagent for GPT measurement. Here, the amino acid is an amino acid serving as a substrate of an enzyme (GOT or GPT) to be measured, and is L-aspartic acid in the case of a GOT measurement reagent and L-alanine in the case of a GPT measurement reagent. Α-KG is the other substrate of the enzyme.

The chelating agent that can be used in the present invention is a polydentate ligand that coordinates a metal ion to give a water-soluble chelate compound, and specifically, ethylenediaminetetraacetic acid (EDTA) and trans-1. , 2-diaminocyclohexanetetraacetic acid (CyDTA), diethylenetriaminepentaacetic acid (DTPA), glycol ether diaminetetraacetic acid (GEDTA), triethylenetetramine hexaacetic acid (T
THA), ethylenediaminediacetic acid (EDDA), ethylenediaminetetrakis (methylenephosphonic acid) (ED
TPO), ethylenediamine dipropionate hydrochloride (E
DDP), hexamethylenediaminetetraacetic acid (HDT
A), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-bis (methylenephosphonic acid) (EDDP
O), nitrilotriacetic acid (NTA), nitrilotris (methylenephosphonic acid) (NTPO), nitrilotriapropionate (NTP), dihydroxyethylglycine (DHE)
G), iminodiacetic acid (IDA) and the like, and salts thereof.

The chelating agent is preferably 0.002 to 50 in a buffer solution in which the reagent constituents (ie, α-KG and substrate amino acid) are dissolved to a desired concentration.
The concentration range is mM, more preferably 0.02 to 50 mM, and most preferably 0.1 to 20 mM.
When the chelating agent concentration is lower than 0.002 mM, α-
This is not preferable because the stability of KG is insufficient. Also, 50
If it exceeds mM, there is no problem in terms of the stability of α-KG, but the measured values of activity of GOT, GPT and the like tend to be low, which is not preferable.

The pH of the α-KG-containing liquid thus prepared can be set to a desired value with a suitable acid (eg HCl) or alkali (eg KOH). The buffer solution used in the present invention is not particularly limited as long as it has a buffering capacity near the target pH. In order to maintain the stability of α-KG alone, the pH may be 4.0 to 9.5. However, in most cases, since it is used by mixing with the other reagent not containing α-KG, the pH of the reaction system at the time of mixing is set in the above-mentioned pH range so as to be set near the target optimum. It may be appropriately adjusted and used.

As the above-mentioned buffer, conventionally known phosphate buffer, acetate buffer, ethanolamine buffer, borate buffer, citrate buffer, tartrate buffer, or zwitterionic buffer, specifically, Is glycine; taurine; glycylglycine; aminoacetamide; N-substituted glycine,
For example, N- (2-acetamido) iminodiacetic acid, N-
Tris (hydroxymethyl) methylglycine, or N,
N-bis (2-hydroxyethyl) glycine; N-substituted taurine, such as 2-morpholinoethanesulfonic acid,
Piperazine-N, N'-bis (2-ethanesulfonic acid), N- (2-acetamido) 2-aminoethanesulfonic acid, N, N-bis (2-hydroxyethyl) -2-
Aminoethanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, cyclohexylpropanesulfonic acid, 3- [N, N
-Bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid, N-tris (hydroxymethyl) methyl-2-hydroxy-3-aminopropanesulfonic acid, or piperazine-N, N'-bis (2- Hydroxypropane sulfonic acid); an aliphatic amine such as bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, or (2-aminoethyl) trimethylammonium chloride hydrochloride; or an N-substituted aminopropane sulfonic acid such as , 3-morpholinoprone sulfonic acid, N-tris (hydroxymethyl) -3-aminopropane sulfonic acid, cyclohexyl propane sulfonic acid and the like are used. In addition to the essential compounding ingredients, if necessary, ingredients generally added to clinical test reagents, for example, preservatives such as azide, and various surfactants can be appropriately added.

The α-K according to the present invention having the above structure
The G liquid reagent can maintain stability for more than half a year. This α-KG-containing reagent is, as described above, the GOT.
Alternatively, it can be used as a reagent for GPT measurement. On this occasion,
As a reagent composition, GOT and GPT are added to the α-KG containing reagent.
G is composed by mixing with another substrate substance (L-aspartic acid or L-alanine) used in the measurement of G
The OT or GPT can be accurately measured.

[0024]

The mechanism of the stabilization of α-KG according to the present invention is not clear at present, so the present invention is not limited to the following reasoning. However, when α-KG and an amino acid are made to coexist in a liquid state, it is a contaminant. It is speculated that the non-enzymatic degradation of α-KG, which is thought to be caused by the (impurity) metal, is effectively suppressed by the chelating agent, and that stabilization is possible for a long period of time. To be done.

[0025]

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 4.6 g of α-KG and 70 g of L-aspartic acid sodium salt were dissolved in 100 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer to make the total amount 1 liter, The pH was adjusted to 7.8 (25 ° C.) with KOH to prepare an α-KG-containing solution for GOT. Separately from this, α-KG 6.8 g and L
-Alanine 90 g was dissolved in 100 mM HEPES buffer to make the total volume 1 liter, and then pH was adjusted to 7.5 with KOH.
The temperature was adjusted to (25 ° C.) to obtain an α-KG-containing solution for GPT. Α-KG-containing solution for GOT and α-for GPT
To the KG-containing solution, EDTA disodium salt was added so as to have concentrations of 0.3 mM, 1.5 mM, 3.0 mM and 15.0 mM, respectively, to prepare an α-KG-containing liquid reagent according to the present invention.

This liquid reagent containing α-KG was treated at 25 ° C at 25 ° C.
The sample was stored at 37 ° C and 37 ° C, and the content of α-KG on the day of storage (the day of preparation), after one month, after three months, and after six months was confirmed by the following operation. That is, 1 ml of each liquid reagent containing α-KG was diluted to 20 ml with 0.1 M triethanolamine buffer (pH 7.6). To 0.1 ml of the diluted solution, 2.9 ml of 0.1 M triethanolamine buffer (pH 7.6) containing NADH (10 mg / ml) and ammonium chloride (160 mg / ml) was added, and then a glutamate dehydrogenase aqueous solution ( 60 mg /
(10 ml) was added, the mixture was reacted at 37 ° C for 10 minutes, and then the absorbance at a wavelength of 340 nm was measured. Separately from this, the same operation was performed using the α-KG standard solution, and the absorbance was measured. Further, the same operation was performed using 0.1 M triethanolamine buffer solution (pH 7.6) instead of the standard solution, and the absorbance was measured to give a blank. From each absorbance, the content of α-KG contained in the storage solution was determined, and the value immediately after the preparation was calculated to be 100% and calculated. The results are shown in Table 1 (α-KG containing solution for GOT) and Table 2 (α-KG containing solution for GPT). It could be confirmed that α-KG in the liquid reagent prepared according to the present invention was stabilized over a long period of time.

[0027]

[Table 1] GOT α-KG-containing solution storage temperature EDTA amount Preparation day 1 month 3 months 6 months 10 ° C storage No additive 100.0% 99.5 99.1 97.5 0.3 mM 100.0% 99.6 99.9 98.9 1.5 mM 100.0% 99.7 99.6 99.0 3.0mM 100.0% 99.6 100.1 99.4 15mM 100.0% 99.9 100.1 100.1 25 ° C Storage No additive 100.0% 99.1 96.8 92.8 0.3mM 100.0% 99.2 99.0 97.7 1.5mM 100.0% 98.9 98.5 97.4 3.0mM 100.0% 99.3 99.1 98.9 15 mM 100.0% 99.0 99.0 98.3 Store at 37 ° C No additive 100.0% 93.9 87.7 73.6 0.3 mM 100.0% 98.9 97.8 97.7 1.5 mM 100.0% 99.0 98.8 97.7 3.0 mM 100.0% 98.9 98.9 98.3 15 mM 100.0% 99.4 99.0 99.0

[0028]

[Table 2] Storage temperature of solution containing α-KG for GPT EDTA amount Preparation day 1 month 3 months 6 months 10 ° C Storage no additive 100.0% 98.5 98.1 94.6 0.3 mM 100.0% 100.0 99.9 99.6 1.5 mM 100.0% 99.9 99.9 99.9 3.0mM 100.0% 100.1 100.0 99.7 15mM 100.0% 99.9 99.8 99.4 Store at 25 ° C No additive 100.0% 95.0 85.5 70.2 0.3mM 100.0% 99.1 99.0 97.9 1.5mM 100.0% 99.8 99.5 99.4 3.0mM 100.0% 100.3 99.8 99.8 15 mM 100.0% 99.7 99.3 98.9 Store at 37 ° C No additive 100.0% 79.0 41.8 11.5 0.3 mM 100.0% 99.4 98.9 97.9 1.5 mM 100.0% 99.2 99.2 99.3 3.0 mM 100.0% 99.7 99.9 98.9 15 mM 100.0% 98.8 97.9 96.0

Example 2 α-KG-containing solution for GOT and G according to Example 1 above
An α-KG-containing solution for PT was prepared, and EDTA was added to the solution.
Dipotassium salt, CyDTA, GEDTA, and NTA were each added to 2 mM, and α-KG according to the present invention was added.
A containing liquid reagent was prepared. This α-KG-containing liquid reagent was stored at 10 ° C, 25 ° C, 37 ° C, and on the day of storage (preparation),
The content of α-KG after 1 month, 3 months and 6 months was confirmed by the same operation as in the above example. From each absorbance, the content of α-KG contained in the storage solution was determined, and the value immediately after the preparation was calculated to be 100% and calculated. The results are shown in Table 3.
(Α-KG-containing solution for GOT) and Table 4 (α-KG for GPT)
KG-containing solution). It could be confirmed that α-KG in the liquid reagent prepared according to the present invention was stabilized over a long period of time. NTA is α-K for GPT
Experiments other than G-containing solution storage at 37 ° C, especially α-for GOT
A sufficient effect could be obtained in the storage experiment of the KG-containing solution.

[0030]

[Table 3] Storage temperature of solution containing α-KG for GOT Chelating agent Preparation day 1 month 3 months 6 months 10 ° C Storage no additive 100.0% 99.5 99.1 97.5 EDTA 100.0% 99.6 99.6 99.1 CyDTA 100.0% 99.9 99.8 99.5 GEDTA 100.0 % 99.9 99.6 99.2 NTA 100.0% 99.9 99.3 98.9 Store at 25 ° C No additive 100.0% 99.1 96.8 92.8 EDTA 100.0% 99.0 98.7 98.1 CyDTA 100.0% 99.8 98.9 98.4 GEDTA 100.0% 99.4 99.4 99.1 NTA 100.0% 99.7 99.1 99.0 37 ° C No additive 100.0 % 93.9 87.7 73.6 EDTA 100.0% 99.3 98.0 97.4 CyDTA 100.0% 99.5 99.0 98.2 GEDTA 100.0% 99.2 98.9 98.5 NTA 100.0% 98.6 97.8 96.4

[0031]

[Table 4] Storage temperature of solution containing α-KG for GPT Chelating agent Preparation day 1 month 3 months 6 months 10 ° C Storage no additive 100.0% 98.5 98.1 94.6 EDTA 100.0% 99.3 99.2 99.2 CyDTA 100.0% 99.8 99.9 99.5 GEDTA 100.0 % 100.0 99.7 99.4 NTA 100.0% 99.9 97.2 96.5 25 ° C storage No addition 100.0% 95.0 85.5 70.2 EDTA 100.0% 98.2 97.5 97.3 CyDTA 100.0% 99.5 98.7 98.4 GEDTA 100.0% 99.0 98.1 97.5 NTA 100.0% 95.9 89.5 78.9 37 ° C storage no addition 100.0 % 79.0 41.8 11.5 EDTA 100.0% 99.4 98.9 97.9 CyDTA 100.0% 99.2 99.2 99.3 GEDTA 100.0% 99.7 99.9 98.9 NTA 100.0% 89.4 53.8 20.7

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, α-KG can be stabilized by making a liquid reagent containing α-KG and an amino acid coexist with a chelating agent.
-A KG-containing liquid reagent can be provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoko Enfuji 1-11-4 Higashikanda, Chiyoda-ku, Tokyo Inside Jatron Co., Ltd. (56) Reference JP-A-57-39800 (JP, A) JP-A-57 -39799 (JP, A) JP-A-59-28497 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12Q 1/00 C12Q 1/52 G01N 33/50 CA / BIOSIS / MEDLINE / W PIDS (STN)

Claims (2)

(57) [Claims] 1. A structure which is divided into an enzyme and a substrate.
A method for stabilizing a substrate reagent in a reagent-based liquid reagent, wherein the substrate reagent is 2-oxoglutaric acid and L-alanine or L- as a substrate for a glutamate pyruvate transaminase or a glutamate oxaloacetate transaminase that is an enzyme to be measured. The method for stabilizing a substrate reagent as described above, which comprises aspartic acid and a coexisting chelating agent. 2. An enzyme and a substrate which are divided into 2
A substrate reagent in a reagent system liquid reagent, wherein the substrate reagent is 2-oxoglutarate and L as substrates for a glutamate pyruvate transaminase or a glutamate oxaloacetate transaminase that is an enzyme to be measured.
-Alanine or L-aspartic acid, together with a chelating agent together, the above-mentioned substrate reagents.