JP3511211B2 - Amylase activity measurement reagent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a reagent for measuring amylase activity. More particularly, it relates to an improved liquid reagent for measuring amylase activity which is stable for a long period of time. [0002] Amylase is a general term for enzymes that hydrolyze starch and includes α-, β- and γ-amylase. The only human amylase is α-amylase, most of which is derived from saliva and pancreatic juice. Other reported organs include lung, liver, kidney, small intestine, myocardium, mammary gland and adipose tissue, but their amylase levels are very low. Wo
Since Hlgemut developed a method for measuring amylase activity in blood and urine, it has been applied clinically and
In 1999, Elman et al. Reported hyperamylaseemia during acute pancreatitis. Since then, it has been applied to diagnosis of ectopic amylase-producing tumors such as lung cancer in addition to parotitis. Conventionally, various methods for measuring amylase activity have been proposed. For example, the following equations (1) and (2)
There is a method for measuring amylase activity by measuring the rate of increase of 2-chloro-4-nitrophenol (hereinafter, sometimes referred to as CNP) produced by the above reaction. [0004] In the above equations (1) and (2), B
-G5-β-CNP is 3-ketobutylidene-β-2-chloro-4-nitrophenylmaltopentaoside, G3-β-CNP is β-2-chloro-4-nitrophenylmaltotrioside, G2-β-CNP is β-2-chloro-4-nitrophenyl maltoside. There is also known a method for measuring amylase activity by measuring the rate of increase of a quinoid dye produced by the reaction of the following formulas (3), (4) and (5). [0007] In the above equations (3) to (5), CM
S is carboxymethyl starch, GA is glucoamylase, GOD is glucose oxidase, POD is peroxidase, and 4AAP is 4-
Aminoantipyrine, ESPT is N-ethyl-N
-Sulfopropyl-m-toluidine. Further, there is also known a method for measuring amylase activity by measuring the rate of increase of CNP produced by the reaction of the following formulas (6) and (7). [0010] In the above equations (6) and (7), G
5-β-CNP is β-2-chloro-4-nitrophenyl maltopentaoside, G3 is maltotriose, and G2-β-CNP is β-2-chloro-4-nitrophenyl maltoside Yes, CNP is 2-chloro-
4-nitrophenol. Further, there is also known a measuring method for measuring amylase activity by measuring the rate of increase of pNP produced by the reaction of the following formulas (8) and (9). [0013] In the above equations (8) and (9), 3
pNP-G7 is p-nitrophenylmaltoheptaside, pNP-G4 is p-nitrophenylmaltotetraoside, pNP-G3 is p-nitrophenylmaltotrioside, and pNP is p-nitrophenol. It is. [0015] Among the various measuring methods described above, the amylase activity measuring method using the reaction system of the formulas (1) and (2) has a clear substrate structure and a stoichiometric amount. Reaction proceeds theoretically, and the reaction product is the optimal pH of amylase.
Since an ionization rate close to 100% can be obtained near 7.0, this is an excellent method that can perform measurement with high sensitivity and good reproducibility. However, as a reagent for measuring amylase activity using this reaction system, a liquid reagent that can be stably stored for a long time has not been developed.
That is, conventionally, most of the reagents have been supplied in the form of a lyophilized body for the main purpose of stability of the reagent components, and the reagent for measurement was prepared and used at the inspection site from the lyophilized body. . However, in order to improve the workability and efficiency at the site of clinical testing, in recent years, in particular, there has been a demand for supplying a reagent in a liquid form that can be directly used in the testing process without requiring a preparation process. However, the enzymes and substrates used in this system can be stably stored in a solution state for a long period of time, and it is extremely difficult to set the reaction pH near the optimum pH of amylase, pH 7.0. I thought it was. That is,
Near the optimum pH of α-amylase, pH 7.0, α
-Glucosidase and β-glucosidase can maintain stability, but cannot be stored for a long time because the substrate B-G5-β-CNP is hydrolyzed, aglycone CNP is generated, and the absorbance is increased. The present inventors have conducted intensive studies to solve these problems, and as a result, in order to stabilize the substrate, (a) a substrate-containing reagent having an acidic pH,
(B) It has been found that the above object can be achieved by dividing into two reagents, that is, an enzyme-containing reagent set so that the pH at the time of the amylase reaction is around 7.0. The present invention is based on these findings. [0017] Accordingly, the present invention provides a three-
Β-2-chloro- produced by amylase using ketobutylidene-β-2-chloro-4-nitrophenylmaltopentaoside (B-G5-β-CNP) as a substrate
4-nitrophenylmaltotrioside (G3-β-C
NP) or β-2-chloro-4-nitrophenylmaltoside (G2-β-CNP) is further converted to 2-chloro-4-nitrophenol (CNP) and glucose by α-glucosidase and β-glucosidase, as described above. The reagent for the method for measuring amylase comprising measuring an increase in the amount of 2-chloro-4-nitrophenol (CNP), wherein at least 3-ketobutylidene-β-2-
Chloro-4-nitrophenylmaltopentaoside (B
-G5-β-CNP) and a second reagent containing at least α-glucosidase and β-glucosidase, and the pH of the first reagent is 4 to 6.5.
(Especially 4.0 to 6.5) and the pH of the second reagent is 6
8 (in particular, 6.0 to 8.0) I der, p of the second reagent
H is the amylase reaction when the second reagent is mixed with the first reagent.
PH is set to be 7, and Ca ²⁺ ion and chloride ion are present in the first reagent and / or the second reagent, and each is a liquid reagent. And a reagent for measuring amylase activity. Hereinafter, the present invention will be described in more detail. The liquid reagent of the present invention is used in a measurement method for determining amylase activity by generating CNP by utilizing the reactions of the above formulas (1) and (2) and measuring the rate of increase. The enzyme and the substrate used in the amylase measurement are kept under stable conditions and the reaction pH is adjusted to the optimal pH of the amylase.
In order to set H at around pH 7.0, a substrate-containing reagent (first reagent) whose pH has been acidified is first prepared. The pH of the substrate-containing reagent is 4 . 0 to pH 6.5 . When the pH is higher than 7, the stability of the substrate is deteriorated. When the pH is lower than 4, the substrate is liable to salt out during storage. In addition, in order to set the pH during the amylase reaction so as to be around pH 7 (particularly pH 7.0), it is preferable to use a buffer having a low buffering capacity as the substrate-containing reagent. The buffer having a weak buffer capacity under acidic conditions is not particularly limited, and any buffer can be used. In addition, a strong buffer solution under acidic conditions can be used as the buffer solution for the substrate-containing reagent.
It is preferable to use as low a concentration as possible (specifically, 0.1 to 100 mM, particularly 1 to 10 mM). Examples of the buffer having a high buffering capacity include a succinate buffer, an acetate buffer, a citrate buffer and a tartrate buffer. B-G5-β contained in the first reagent
-CNP can be appropriately set depending on the reaction conditions, but may be at least 1.0 mM or more, preferably 3.0 mM.
M or more. In the present invention, further, an enzyme-containing reagent (second reagent) set so that the pH at the time of amylase reaction when mixed with the above-mentioned substrate-containing reagent (first reagent) becomes near pH 7 (particularly pH 7.0). ) Is prepared. Since α-glucosidase and β-glucosidase, which are enzymes used for the amylase activity measurement, are stable near neutrality, the pH of the enzyme-containing reagent itself is also set to around pH 7. Here, the substrate-containing reagent (first reagent) has a low buffering capacity, but is acidic, so that the enzyme-containing reagent (second reagent) has a pH of 7 (particularly p
A buffer solution having a buffer capacity around H7.0) is used. Specifically, pH 6 to pH 8 (particularly pH 6.0 to pH 8.0).
0), preferably pH 6.5-7.5. This pH
Outside this range, α-glucosidase and β-glucosidase become unstable. Buffers that can be used in the enzyme-containing reagent (second reagent) include piperazine-N, N ′
Bis (2-ethanesulfonic acid) buffer, phosphate buffer,
Various good buffers and the like can be mentioned. Examples of the enzymes used in the second reagent of the present invention is not particularly limited, for example, microorganisms [e.g., whip <br/> Caro micelle scan (Saccharomyces s)] α- glucosidase or derived, sweet almond (Sweet almond). The added amount of these enzymes is 500 U or more, preferably 1000
What is necessary is just to select and use suitably 10000-10000U. In the reagent of the present invention, calcium ion as an amylase protecting agent and chloride ion as an α-amylase activator are simultaneously or separately added to both or one of the first and second reagents. Must exist. Calcium ions and chloride ions can be contained in any form. For example, calcium chloride, sodium chloride, and / or potassium chloride are preferably used. 100 mM, preferably 1.0-100 m
It can be used in the range of M. To the reagent of the present invention, in addition to the above essential components, generally added components such as a chelating agent such as EDTA, a preservative such as azide, and various surfactants are appropriately added. Can be used. When the amylase activity measuring reagent of the present invention thus constituted is used, if the test sample contains amylase, B-G5-β-CNP ( G3-β-CNP or G2-β-CNP in combination with the action of calcium ions (amylase enzyme protectant) and chloride ions (amylase enzyme activator)
Is hydrolyzed to Furthermore, G3-β-CNP or G2-
β-CNP is finally converted to glucose and C by the coupling enzymes α-glucosidase and β-glucosidase.
Hydrolyzed to NP. The amylase activity can be determined by measuring the rate of increase of CNP produced in this reaction by a usual method. Further, the liquid reagent of the present invention can be stored for a long period of time, and has a great effect in the field of clinical examination. EXAMPLES The present invention will now be described specifically with reference to examples, but these examples do not limit the scope of the present invention. Example 1 Stability of Substrate (1) Preparation of Reagents The pH values were 3.5, 4.0, 5.0, 6.0,
6.5, 7.0, and 7.5 of each 10 mM succinic acid-N
BG-β-CNP (3.7 mmol) and sodium chloride (50 mmol) were dissolved in an aOH buffer to prepare a first reagent in a total volume of 1 liter. Further, α-glucosidase derived from Saccharomyces (6000 U), β-glucosidase derived from Sweet almond (10000 U), EDTA2Na (0.5 mmol), and calcium chloride dihydrate (1.0 mmol) were added in an amount of 0.1%. A second reagent was prepared by dissolving in 1M piperazine-N, N'-bis (2-ethanesulfonic acid) buffer (pH 7.1) and making the total amount 1 liter. Each of these reagents was stored at 10 ° C., and on the day of storage, reagent stability at 1 week, 2 weeks, 3 weeks, 1 month, 2 months and 3 months was confirmed by the following test. (2) Test operation 200 μl of the second reagent was added to 5 μl of physiological saline,
After heating at 7 ° C. for 5 minutes, 200 μl of each first reagent was added, and the absorbance at a wavelength of 405 nm after heating at 37 ° C. for 5 minutes was measured. The results are shown in FIG. As is clear from FIG. 1, the first reagent (substrate-containing reagent) maintained in the pH range of 4 to 7 shows good stability and can be used as an amylase measurement reagent. If the pH exceeds the above range, the initial absorbance increases with the extension of the storage period, so that it becomes unsuitable as a reagent for measuring amylase for long-term storage. Example 2 Measurement of α-Amylase Activity (1) Preparation of Reagent Reagents were added to 10 mM succinate-NaOH buffers at pH 5.0.
BG-β-CNP (3.7 mmol) and sodium chloride (50 mmol) were dissolved to prepare a first reagent in a total volume of 1 liter. The second reagent was prepared in the same manner as in Example 1. Each of these reagents was stored at 10 ° C., and on the day of storage, one month, two months, and three months later,
Amylase activity was measured by the following test procedure. (2) Test procedure 5 μl of the control serum with a known amylase activity value was added to the second reagent 20
After adding 0 μl and heating at 37 ° C. for 5 minutes, 200 μl of the first reagent was added and heated at 37 ° C., and the change in absorbance at a wavelength of 405 nm for 3 to 5 minutes after the addition of the first reagent was determined. Amylase activity was determined from the following equation. Table 1 shows the results. [Equation 5] 0.0165 in the formula is a molar molecular absorbance coefficient of CNP. [Table 1] On the day of storage 1 month 2 months 3 months after administration Serum A (Amirase activity = 79 U / l) 72 U / l 74 U / l 73 U / l 76 U / l Serum control B (Amirase II) (Activity = 170 U / l) 170 U / l 170 U / l 174 U / l 172 U / l As is clear from Table 1, the reagent according to the present invention shows a decrease in reagent performance even after long-term storage. Is not observed, and the amylase activity in the sample can be accurately measured. The reagent for measuring amylase of the present invention can be stored in a liquid state in a dark place or a light place at room temperature or at a low temperature for a long period of time (at least half a year or more). . Therefore, it can be stored for a long time at the site where the clinical test is performed, and can be applied to an automatic analyzer or the like without using a dissolution operation when used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the storage stability of first reagents of various pHs prepared in Example 1.

Continuation of the front page (56) References JP-A-5-123193 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-3/00 EUROPAT (QUESTEL) JICST file (JOIS) BIOSIS / MEDLINE / WPID S (STN) CA (STN)

Claims (1)

(57) [Claims] (Claim 1) 3-Ketobutylidene-β-2-chloro-
Β-2-chloro-4-nitrophenylmaltotrioside or β-2-chloro- produced by amylase using 4-nitrophenylmaltopentaoside as a substrate
4-Nitrophenyl maltoside is further treated with α-glucosidase and β-glucosidase to give 2-chloro-4-
The reagent for the amylase assay comprising measuring the increase in the amount of 2-chloro-4-nitrophenol instead of nitrophenol and glucose, wherein at least 3-ketobutylidene-β-2-chloro-4-nitro A first reagent containing phenylmaltopentaoside, and a second reagent containing at least α-glucosidase and β-glucosidase, wherein the pH of the first reagent is 4 to 6.5, and the pH of the second reagent is What but 6-8 der,
The pH of the second reagent is determined by mixing the second reagent with the first reagent.
The pH is set to 7 during the amylase reaction.
A reagent for measuring amylase activity which is stable for a long time , wherein Ca ²⁺ ion and chloride ion are present in the first reagent and / or the second reagent, and each is a liquid reagent.