JPS6131098A - Reagent for determining choline esterase - Google Patents

Abstract

PURPOSE:To improve the stability of the titled reagent for determining choline esterase using acetylcholine as a substrate, by keeping the first reagent consisting of acetate kinase, adenosine triphosphate, etc. and the second reagent of acetylcholine respectively at specific pH values. CONSTITUTION:A reagent for determining choline esterase, obtained by incorporating the first reagent, prepared by dissolving acetate kinase, adenosine triphosphate and reduced form beta-nicotinamide adenine dinucleotide as principal components in a buffer solution, and adjusting the pH of the resultant solution to 8-10 with the second reagent prepared by dissolving acetylcholine as a principal component in a buffer solution, and adjusting the pH of the resultant solution to 3.5-5.5 at a specific ratio in measuring the choline esterase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides cholinesterase (hereinafter abbreviated as ChE)
This relates to quantitative reagents.

ChE quantification methods are currently used in clinical laboratories.

(1) A method of measuring pH changes using acetylcholine as a substrate.

(2) SH released using thiocholine ester as a substrate
How to measure groups.

(3) A method that uses benzoylcholine as a substrate and uses choline oxidase to measure the liberated choline as Hz (h).

and so on. Among these methods, method (1) is a standard method that has been used in laboratories for a long time, but it is difficult to maintain optimal po, and the pH change does not necessarily parallel the degree of discoloration of the indicator. It is bad to arrive at the coal <
, <21. (Currently, it is being replaced by the sl method. However, in (2) there are errors due to coexisting SH compounds, and in (3) there are problems such as various problems related to H0□ determination and interference by phenol in the coloring system. However, none of them have reached the point where we are satisfied at present.

From this point of view, JP-A-58-155099 proposes a ChE quantitative reagent containing acetylcholine, acetate kinase, and adenosine triphosphate. To explain this reagent, acetylcholine is used as a substrate, and when ChE in a biological sample acts, the substrate is decomposed into acetic acid and choline.

When acetate kinase acts on this, acetic acid is converted to acetyl phosphate using adenosine diphosphate (hereinafter referred to as ATP) as a cosubstrate. This reaction formula is shown below.

+Adenosine diphosphate The action of acetate kinase has substrate specificity only for acetic acid, and does not work even when other organic acids, inorganic acids, etc. are present. Next, the determination of ChE is completed by quantifying the acetyl phosphate or adenosine diphosphate (hereinafter referred to as ADP) produced here. At this time, acetyl phosphate can be quantified by visible colorimetry by reacting with an appropriate chromogen, and ADP can be determined by using a coupled enzyme system of pyruvate kinase and lactate dehydrogenase. Quantifiable. In particular, the latter method is an extremely advantageous method in which the entire reaction system is an enzyme system and is not affected by coexisting substances.The reaction formula in this case is shown below.

ADP + Phosphoenolpyruvate Pyruvate Kinase ---> ATP + Pyruvate (However,
NADH represents reduced β-nicotinamide-adenine dinucleotide, and NAD+ represents oxidized β-nicotinamide-adenine dinucleotide. ) By measuring the absorbance of this NADH in the ultraviolet region (340 nm), highly accurate quantification can be performed extremely easily. In addition, since ADP can be regenerated by the action of pyruvate kinase, the action of acetate kinase on acetate is enhanced, making extremely sensitive measurement possible.

How to actually commercialize this reagent.

A reagent containing acetate kinase, ATP, and NADH as the main components (hereinafter referred to as the first reagent) and a reagent containing the substrate acetylcholine as the main component (hereinafter referred to as the second reagent) were prepared respectively, and the ChE measurement was performed. Both reagents are mixed.

The po of the first reagent is 7.0-8.0, which is the optimum pH of ChE.
The pH of the second reagent is not particularly controlled and is around neutral.

In this case, in the first reagent, the absorbance at 340n+w derived from NADH tended to decrease during reagent storage, and in the second reagent, spontaneous decomposition of acetylcholine tended to occur during storage. . When this natural decomposition of acetylcholine occurs, acetic acid is produced, and when the first reagent and the second reagent are mixed, the reaction proceeds, 34
The absorbance at 0 nm will decrease.

That is, the measurement principle of the above reagent is ultimately 340 nm.
Because it measures the rate of decrease in absorbance at
It is desired that the absorbance of the first reagent when the first reagent and the second reagent are mixed and the absorbance when the first reagent and the second reagent are mixed are at least 1.0, and the absorbance at 340 nm is Since 2.0 is the limit due to the performance of a normal spectrophotometer needle, the period during which the absorbance of 2.0 decreases to around 1.0 is the period of use of the reagent after preparation.

This point is not sufficient, and there is a desire for a reagent that can be prepared once and used for as long as possible.

As a result of extensive research to satisfy these requirements, the present inventors have found that by controlling the pH of each of the first and second reagents within a specific range, the stability of both reagents after preparation can be improved. The present invention was achieved by discovering that this can be dramatically improved.

That is, two aspects of the present invention include acetate kinase, ATP, and NAD.
It consists of a first reagent containing H as a main component and a second reagent containing acetylcholine as a main component, and the pH of the first reagent is 8.0.
~10.0, and the second reagent has a pH of 3.5 to 5.5.

The reagent of the present invention is configured as follows, for example.

+11 Enzyme reagent (lyophilized product) Acetate kinase 3 Pyruvate kinase, lactate dehydrogenase, ATP, phosphoenolpyruvate, NADH (2
) Substrate agent (powder) Acetylcholine, ammonium sulfate (3) Enzyme reagent solution buffer, magnesium salt, potassium salt, preservative (4) Substrate agent solution buffer, preservative For preparing the first reagent and second reagent is (1) with (3).

This can be done by dissolving (2) in (4) <,C
When hE measurement is specified, the first reagent and the second reagent may be mixed at a predetermined ratio.

In the present invention, it is necessary to control the pH of the first reagent and the second reagent after the preparation of the former to 8.0 to 10.0 and the latter to 3.5 to 5.5.

When deviating from this pH, in the first reagent.

The absorbance at 340r++++ significantly decreased, and a large amount of spontaneous decomposition of acetylcholine was observed in the second reagent. The po of this first reagent is 8.0-1
In order to control the pH of the second reagent to 3.5 to 5.5, for example, the following buffer can be used. As a buffer used in the first reagent.

For example, tris(hydroxymethyl)aminomethane-II
cL) Lis(hydroxymethyl)aminomethane-maleic acid, N-2-hydroxyethylpiperazine-N゛-
Ethanesulfonic acid, NaOH, glycine-Mo1. ) Liethanolamine hydrochloric acid-NaOH, etc., as long as the range of normal use is p)I e, o to 10.0.

Buffers used in the second reagent include 1, for example, potassium hydrogen phthalate-)ICI, citric acid-sodium citrate, maleic acid-NaOH+glycine-HCl
, 3.3'-dimethylglutaric acid-NaOH, lactic acid-sodium lactate, etc. The usual range of use is pH 3.5 to 5.
It is sufficient if it has a value of 5°.

The amount of other reagents used is acetylcholine 20~
200mM, acetate kinase 5-100 u/ml, A
TP 1.5-15a+M, pyruvate kinase 3
~50 u/ml, lactate dehydrogenase 1-20 u/ml
, NADHO, 1-1.0mM, NADHO11-
Approximately 1° is appropriate.

Next, in order to quantify ChE, after mixing the first reagent and the second reagent at a certain ratio, the optimal p of ChE, which is the target substance, is
The first reagent and the second reagent may be mixed so that H is 7.0 to 8.0.

At that time, the liquid ratio of the first reagent and the second reagent varies depending on the autoanalyzer used, but preferably the first reagent and the second reagent are z:i -10:l (volume ratio,
same as below. ) and 9 more preferably 2:1 to 8:1
The ratio is most preferably 2:1 to 5:1. Also,
The concentration of the buffer for the first reagent and the second reagent is 9. For example, the liquid ratio of the first reagent and the second reagent is 2:1 to 5:1. The pH of the first reagent is 8.0 to 10.0. The pn of the second reagent is 3.5-5
．． 5, and if the condition is set that the pH when the first reagent and the second reagent are mixed is between 7.0 and 8.0, N can be determined by a simple experiment. for example,
The first reagent is tris(hydroxymethyl)aminomethane.
When maleic acid is used as the second reagent and citric acid-NaOH is used, the former is 25-70mM and the latter is 90-130mM.
And it is sufficient.

Next, one embodiment of the present invention will be specifically explained using examples.

Example 1 +l) Enzyme reagent (lyophilized product) Acetate kinase 2X10' unit pyruvate kinase lX10' unit lactate dehydrogenase
1×103 Uni 7) ATP
1 gr phosphoenolpyruvate
0.05 grNADH0,14gr (2) Substrate agent (powder) Acetylcholine 1.4 gr Ammonium sulfate 0.33 gr (3) Enzyme reagent solution Tris(hydroxymethyl)aminomethane-) IcI
(ρ) 19.0) 30 mM/L magnesium sulfate 15 mM/L potassium chloride
50 mM/L sodium azide
10 +IIM/L (4) Substrate side solution maleic acid-NaOH (pH 4,5) 95 m
M/L sodium azide 5mM/L The first reagent was prepared by dissolving the enzyme reagent (1) in 100 ml of the enzyme reagent solution of (3), and the substrate agent (2) was added to the substrate of (4). A second reagent was prepared by dissolving with 25+sl of the side lysis solution. Both reagents were left in a thermostat at 10° C. for 7 days, and the absorbance of the first reagent at 340 nm was compared with that immediately after preparation.

In addition, when the first reagent and the second reagent were mixed at a liquid volume ratio of 4:1 after being left for 7 days, the decrease in the initial absorbance at 340 nm due to acetic acid caused by the decomposition of acetylcholine was measured,
A comparison was also made with the one immediately after preparation.

The results are shown in Table 1.

In addition, the p)I of the first reagent and the second reagent after 11 production is 9 and 0.4.5, respectively, which are the pH of the buffer of the dissolution solution, and the pH after mixing the three reagents is 7.5. Ta.

Table 1 Example 2 The buffer for the enzyme reagent solution was tris(hydroxymethyl)aminomethane-maleic acid pH 8,55-30III.
In addition, the buffer for the solution of the substrate agent was added to glycine-11CI.
The same stability test as in Example 1 was conducted using all the same reagents as in Example 1 except that pH 4.0-75+++M was used, resulting in the results shown in Table 2.

Table 2 The pH of the first reagent and the second reagent after preparation was 8.5.4.0, which is the pn of the buffer of the respective solution, and the pH after mixing both reagents was 7.3. .

Comparative Example 1 The buffer for the enzyme reagent solution was N-2-hydroxyethylpiperazine-No-ethanesulfonefil-NaOHp.
The same stability test as in Example 1 was conducted using the same reagents as in Example 1 except that H7,5-100 mM was used and the substrate agent solution was an aqueous solution without using a buffer. The results are shown in Table 3.

Note that the pH of the first reagent is 7.5, and the pH of the second reagent is 7.5.
．． 0 and 98 after mixing both reagents became 7.5.

Table 3 As shown above, the reagent of the present invention has dramatically improved stability after preparation, and can be stored for a long period of time.

Patent applicant: Unitika Co., Ltd. VC company Yatron Co., Ltd.

Claims (1)

[Claims] (1) Acetate kinase, adenosine triphosphate, reduced β-
It consists of a first reagent whose main component is nicotinamide adenine dinucleotide and a second reagent whose main component is acetylcholine, and the pH of the first reagent is 8.0 to 10.0, and the pH of the second reagent is 8.0 to 10.0. A reagent for quantifying cholinesterase, which has a pH of 3.5 to 5.5.