JPH0573400B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a reagent for quantifying cholinesterase (hereinafter abbreviated as ChE). The methods for quantifying ChE currently used in clinical laboratories include: (1) A method that measures PH changes using acetylcholine as a substrate. (2) Release using thiocholine ester as a substrate
How to measure SH groups. (3) A method using benzoylcholine as a substrate and measuring the liberated choline as H ₂ O ₂ using choline oxidase. 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 not possible to maintain the optimum pH, and the pH change does not necessarily parallel the degree of discoloration of the indicator. Currently, methods (2) and (3) are being replaced because of their poor accuracy. However, in (2), coexisting SH
There are errors due to compounds, problems related to H ₂ O ₂ quantification in (3), and interference by phenol in the coloring system, and so far none of them has been achieved satisfactorily. From this viewpoint, JP-A-58-155099 proposes a ChE quantitative reagent containing acetylcholine, acetate kinase, and adenosine triphosphate. This reagent has excellent accuracy and high performance as a reagent, such as being unaffected by blood components. However, samples with high ChE activity values (serum)
In a so-called serum dilution test, we measure the ChE activity value of a dilution series created by sequentially diluting this with physiological saline, and examine the correlation between the obtained ChE activity value and the dilution rate of serum. A phenomenon in which the correlation deviates from the range of ChE activity values from around 4000 U/L to above is observed, and in this case, the sample must be diluted and measured again. Additionally, the upper limit of the normal range for this reagent at 30°C is approximately 3000U/
However, since it is generally said that the upper limit of the measurement range is about twice the upper limit of the normal value, there is a strong demand for a measurement range of up to about 6000 U/L. In view of these circumstances, the present inventors conducted further intensive research and found that acetyl phosphate, which is accumulated without being consumed during ChE activity measurement, has an adverse effect. The present invention was achieved based on the discovery that when phosphotransacetylase and coenzyme-A are included in order to scavenge phosphoric acid, the upper limit of the measurement range of the reagent increases dramatically. That is, the present invention provides acetylcholine as a substrate, adenosine triphosphate and acetate kinase for converting acetic acid produced by the action of cholinesterase into adenosine diphosphate and acetyl phosphate, and reducing the converted adenosine diphosphate. It is characterized by consisting of pyruvate kinase and lactate dehydrogenase to lead to a decrease in the absorption of type nicotinamide-adenine dinucleotide, and phosphotransacetylase and coenzyme-A to scavenge the converted acetyl phosphate. This is a reagent for quantifying ChE. To explain the reagent of the present invention, when acetylcholine is used as a substrate and ChE in a biological material is reacted with, the substrate is decomposed into acetic acid and choline. When acetate kinase acts on this, acetic acid is converted to acetyl phosphate using adenosine triphosphate (hereinafter referred to as ATP) as a cosubstrate. This reaction formula is shown below. Acetylcholine + H ₂ OChE ―――→ Acetic acid + choline …… Acetic acid + ATP acetate kinase ――――――――→ Acetyl phosphate + adenosine diphosphate …… Next, the acetyl phosphate or adenosine diphosphate produced here (hereinafter referred to as ADP), the quantification of ChE is complete. When quantifying acetyl phosphate produced by the reaction formula, when coenzyme-A and phosphotransacetylase are allowed to interact, it is converted to acetyl coenzyme-A, and the amount produced is measured under ultraviolet light (233 nm).
This can be done by measuring the absorbance of phosphoric acid or quantifying the produced phosphoric acid using a conventional method.
The reaction is shown below. Acetyl phosphate + coenzyme-A phosphotransacetylase――――――――――――――――→ Acetyl coenzyme-A + phosphoric acid……Measurement using the above reaction formula is unstable in the ultraviolet region. Methods for quantifying phosphoric acid have disadvantages such as having to measure at a wavelength (233 nm) or being affected by phosphoric acid present in serum, and are not common. On the other hand, ADP produced by the reaction formula can be measured using a coupled enzyme system of pyruvate kinase and lactate dehydrogenase according to the reaction formula below. The reaction formula in this case is shown below. ADP + Phosphoenolpyruvate Pyruvate Kinase――――――――――――→ ATP + Pyruvate …… Pyruvate + NADH Lactate dehydrogenase――――――――――→ Lactic acid + NAD ⁺ …… (However, NADH is reduced β-nicotinamide
Adenine dinucleotide, NAD ⁺ , is the oxidized β-
Represents nicotinamide-adenine dinucleotide. ) The method shown in the above reaction formula is a method of reducing ultraviolet absorption (340 nm) that is not easily affected by coexisting substances in serum, so it is preferable as a method for measuring cholinesterase.A reagent for quantifying cholinesterase based on this measurement principle is Highly accurate quantification can be performed extremely easily. Furthermore, the action of pyruvate kinase allows the generated ADP to be regenerated into ATP, which enhances the action of acetate kinase on acetic acid, making it possible to perform measurements with extremely high sensitivity. Furthermore, in the present invention, in order to eliminate acetyl phosphate produced by the reaction formula, phosphotransacetylase shown in the reaction formula and coenzyme-A are allowed to coexist in the reaction system. Acetyl coenzyme-A produced in this reaction is
It has absorption in the ultraviolet region, but its maximum absorption is
233 nm, and does not have any adverse effect on this measurement system that measures the decrease in ultraviolet absorption at 340 nm due to NADH. Furthermore, the generated phosphoric acid does not have any adverse effect on this measurement system. This makes it possible to significantly improve the performance of a reagent for quantifying cholinesterase using a coupled enzyme system of acetate kinase, pyruvate kinase, and lactate dehydrogenase. Phosphotransacetylase as used in the present invention is a common name, and its formal name is acetyl coenzyme-A; orthophosphate transferase (EC2.3.1.8). As the phosphotransacetylase for carrying out the present invention, various types derived from microorganisms can be used, including Bacillus stearothermophilus, Streptomyces thermoaceticum, Achromobacter -V-2
It is advantageous to use thermostable phosphotransacetylases produced by thermophilic bacteria such as .
Among these, those derived from Bacillus stearothermophilus are the most preferred because of ease of purification and high specific activity. The reagent of the present invention includes, for example, the reagent described in JP-A-58-155099 containing phosphotransacetylase and coenzyme-A. ml, preferably 5 to 50 U/ml. In addition, coenzyme-A is 0.005 to 1
It may be mA, preferably 0.01 to 0.5mM.
As for the amount of other reagents used, acetylcholine
20-200mM, acetate kinase 5-100U/ml,
ATP 1.5-15mM, pyruvate kinase 3-
50U/ml, lactate dehydrogenase 1-20U/ml, phosphoenolpyruvate 0.1-2mM, NADH 0.1-1
About mM is appropriate. The reaction temperature of the present invention is preferably a temperature used in ordinary clinical chemistry tests, and for example, a temperature of 30° C. can be conveniently used. Furthermore, in carrying out the present invention, all of the above reagent components can be used as one reagent without any problem, but in order to stabilize the reagent components, acetate kinase, pyruvate kinase, lactate kinase, Dehydrogenase, phosphotransacetylase, ATP, phosphoenolpyruvate,
a first reagent containing NADH, a magnesium salt;
It is desirable to use the reagent in combination with a second reagent containing acetylcholine and coenzyme-A.
At that time, the pH of each is 8.0 to 10.0 for the stability of absorbance at 340 nm derived from NADH in the first reagent, and PH8.0 to 10.0 for the stability of acetylcholine and coenzyme-A in the second reagent. It is desirable that the pH is between 3.5 and 5.5. Furthermore, it is desirable to set the buffer concentrations of the first and second reagents so that when the first and second reagents are mixed at a predetermined ratio, the optimum pH for ChE is PH7.0 to 8.0. Examples of the buffer used in the present invention in the first reagent include tris(hydroxymethyl)aminomethane-HCl, blis(hydroxymethyl)aminomethane-maleic acid, and N-2-hydroxyethylpiperazine-N'-ethane. Sulfonic acid-
For NaOH, glycine-KOH, triethanolamine hydrochloride-NaOH, the normal usage range is PH8.0 ~
It should be 10.0. In addition, examples of the buffer used in the second reagent include potassium hydrogen phthalate-HCl, citric acid-
Sodium citrate, maleic acid-NaOH, glycine-HCl, 3,3'-dimethylglutaric acid-
NaOH, lactic acid-sodium lactate, etc., which normally have a pH range of 3.5 to 5.5, may be used. Next, the present invention will be specifically explained using examples. Example 1 ChE consisting of the first reagent and second reagent shown in Table 1
Prepare a quantitative reagent and perform a serum dilution test using standard serum with a ChE activity value of 6500 U/L as the stock solution (standard serum was diluted with physiological saline, and samples with different concentrations of 2/10 were prepared). I went. For the reagent, mix 2.4ml of the first reagent and 0.6ml of the second reagent.
The buffer concentration was set so that the final reaction pH was 7.5. To measure ChE, add 0.02 ml of standard serum sample of each dilution to 2.4 ml of the first reagent, and
After incubating for a minute, 0.6 ml of the second reagent was added to start the ChE reaction, and the decrease in absorption of NADH at 340 nm was monitored over a period of 3 minutes. ChE activity is determined by the rate of absorption decrease at 340 nm per minute.
ChE activity was calculated using the usual calculation method. The results are shown in Figure 1, up to 6500U/L.
A clean linear relationship was obtained between the ChE activity measurements and the sample dilution series. This shows that the measurement range of the reagent according to the present invention is up to 6500 U/L.

[Table] Comparative Example 1 A reagent was prepared excluding phosphotransacetylase in the first reagent and coenzyme-A in the second reagent shown in Table 1, and a serum dilution test was performed using the same sample as in Example 1. I went there. The second result is
As shown in the figure. From Figure 2, a linear relationship can only be obtained between the ChE activity measurement value and the sample dilution series up to about 4000 U/L, and the reagent according to Comparative Example 1 has a performance in the measurement range up to 4000 U/L. It shows. Example 2 Each reagent component shown in Example 1 was prepared as one reagent. The concentrations of each reagent composition component are as follows. Tris (hydroxymethyl) 80mM aminomethane-maleate buffer (PH7.5) Magnesium sulfate 10mM Potassium chloride 25mM Ammonium sulfate 10mM ATP 5mM Phosphoenolpyruvate 1mM NADH 0.26mM Coenzyme-A 0.01mM Acetate kinase (Bacillus stearothermophilus) Origin) 30U/ml Pyruvate kinase (derived from rabbit muscle) 10U/ml Lactate dehydrogenase (derived from rabbit muscle) 5U/ml Phosphotransacetylase (derived from Bacillus stearothermophilus) 10U/mM Acetylcholine chloride 60mM This reaction reagent After incubating 3.0ml at 37℃ for 5 minutes,
Add 0.02 ml of the serum sample prepared in Example 1,
Start the ChE reaction and continue for 3 minutes.
The decrease in absorption of NADH at 340 nm was followed.
As a result of a serum dilution test similar to that in Example 1, results similar to those shown in FIG. 1 were obtained.

[Brief explanation of the drawing]

FIGS. 1 and 2 are diagrams showing the relationship between the dilution factor of the standard serum and the ChE activity value, where the horizontal axis shows the dilution factor of the standard serum and the vertical axis shows the ChE activity value.

Claims (1)

[Claims] 1. Acetylcholine as a substrate, adenosine triphosphate and acetate kinase for converting acetic acid produced by the action of cholinesterase into adenosine diphosphate and acetyl phosphate, and the converted adenosine diphosphate. It is composed of pyruvate kinase and lactate dehydrogenase to reduce the absorption of reduced nicotinamide-adenine dinucleotide, and phosphotransacetylase and coenzyme-A to scavenge the converted acetyl phosphate. Characteristic cholinesterase quantitative reagent. 2. The reagent according to claim 1, wherein the phosphotransacetylase is a thermostable phosphotransacetylase.