JP2002238598A - Composition for calcium ion assay and assaying method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a simple, high-precision and stable reagent for calcium ion determination which is obtained by adding a chelating agent of glycol ether diamine tetraacetate and diethylene triamine pentaacetate and magnesium ions to a calcium ion determination reagent using an activation reaction of phospholipase D with calcium ions and is not influenced by divalent cations except calcium ion. SOLUTION: The method for calcium ion determination comprises treating a liquid to be tested with phospholipase D treated with the chelating agent, a substrate for phospholipase D and phospholipase D and assaying enzyme activity of phospholipase D. The composition for the calcium ion determination is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent composition for measuring calcium ions contained in a subject and a measuring method using the reagent composition.

[0002]

2. Description of the Related Art Calcium ions contained in serum or urine provide useful information in the diagnosis of various diseases, and their measurement is frequently performed in the field of clinical testing. Examples of various diseases include hypocalcemia, such as hypoproteinemia, hypophosphatemia, nephropathy, nephrosis, vitamin D deficiency, hypoparathyroidism, cull disease, and hypercalcemia. , Bone tumor, Addison's disease, emphysema, hyperparathyroidism, renal failure and the like. As a method for measuring calcium ions in biological components,
The O-CPC (orthocresolphthalein comblexon) method is widely used on a daily basis. This method uses O-
This is a color-forming method based on the principle that CPC reacts with calcium ions under alkaline conditions to produce a red-purple dye, but lacks specificity such as being affected by magnesium ions, and is affected by proteins and temperature. There are disadvantages.

[0003] Therefore, in recent years, various enzyme methods having higher specificity have been reported. Each of these methods is as follows, utilizing the phenomenon activated by the calcium ion of the enzyme. (1) Use of phospholipase D as described, for example, in JP-A-62-195297; a composition comprising lecithin, phospholipase D and choline oxidase, JP-A-4-187098; choline-based phospholipid, phospholipase D , A composition comprising choline oxidase, a surfactant and a divalent metal salt, JP-A-4-23999; a composition comprising a non-natural phosphatidylcholine derivative, phospholipase D and choline oxidase or choline dehydrogenase; 170999;
Calcium ion measurement reagent containing a chelating agent.

(2) As a method using phospholipase A2, for example, JP-A-1-231896; a method for measuring phospholipase A2 enzyme activity using phosphorylcholine thioester as a substrate, JP-A-5-168498
Patent Document 1: A method of measuring phospholipase A2 enzyme activity using choline phospholipid or ethanolamine phospholipid as a substrate. (3) As a method using calmodulin, for example, JP-A-62-36199. (4) As a method using pyruvate kinase, for example, JP-A-2-142498. (5) As a method using amylase, see, for example,
276597. (6) As a method using a transaminase, for example, JP-A-5-219992.

In these methods, (1) is problematic in that it is affected by divalent cations other than calcium ions, and the problem is that the enzyme is destabilized by a chelating agent as an activity regulator. (3) has a problem that it is difficult to obtain calmodulin and a problem that the reaction time is long because two-stage activation is used. (4) utilizes the inhibitory action of calcium ions, and thus has a problem in measurement accuracy. In addition, (5) has a disadvantage that it is easily affected by endogenous pancreatic amylase, salivary amylase and the like contained in the sample. Further, (6) is that there is no highly specific method for quantifying hydroxamic acid, which is one of the products, and the effect of endogenous ammonia in the sample is determined when quantifying the other product, ammonia. There are problems such as receiving.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a high specificity for calcium ions in a specimen based on the activation reaction of phospholipase D by calcium ions,
An object of the present invention is to provide a highly accurate and simple measurement method and a reagent composition for measuring calcium used in the method.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made to achieve the above object, and as a result of various studies, the present inventor has found that the specificity for calcium ions can be dramatically increased. Attention was first paid to a chelating agent as a phospholipase D activity regulator. Ethylenediaminetetraacetic acid (EDT) as a chelating agent used in biochemical reactions
Although A) is widely used, this chelating agent treatment also reacts with magnesium ions, and accurate measurement of calcium ions was impossible (see FIG. 1). However, phospholipase D was converted to glycol ether diamine tetraacetic acid (GEDTA). ) And diethylenetriaminepentaacetic acid (DTPA) treatment to increase the specificity for calcium ions among divalent cations such as calcium ions, magnesium ions, manganese ions, nickel ions, cobalt ions, copper ions, and zinc ions. It was found that calcium ions can be accurately quantified when a sample such as serum was used. Furthermore, treatment of phospholipase D with a chelating agent destabilizes the enzyme, and there was a problem with long-term storage as a reagent for quantification. However, it was found that the coexistence of magnesium ion enhances the storage stability of the reagent. The present invention has been completed.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the constitution and preferred embodiments of the present invention will be described in more detail. Phospholipase D used in the present invention is an enzyme derived from cabbage, streptomyces sp (described in JP-A-2000-270857), and Nocardia sp. (Described in JP-A-60-164483) in which calcium ion is essential for the enzyme reaction. However, a microbial enzyme derived from Streptomyces cromofuscus is preferred from the viewpoint of stable supply and stability.

[0009] The substrate of phospholipase D used in the present invention is not particularly limited as long as it produces a reaction product which can be converted into spectroscopic detection after enzymatic reaction with phospholipase D, such as phosphatidylcholine, phosphatidyl. Phospholipids such as glycerol, lysophosphatidylcholine, and lysophosphatidylglycerol, and paranitrophenylphosphorylcholine can be used. Above all, the product of the phospholipase D reaction can be easily and spectroscopically detected with phosphate monoesterase. Preferred is paranitrophenylphosphorylcholine, which has a chromogenic group, is easily available, is water-soluble, and has good stability. When using phospholipids such as phosphatidylcholine, phosphatidylglycerol, lysophosphatidylcholine, and lysophosphatidylglycerol, choline, which is a product of phospholipase D, is oxidized using choline oxidase, and the generated hydrogen peroxide is colored by peroxidase action. By doing so, calcium ions can be quantitatively determined.

The measurement principle of the present invention is shown by the following formula when paranitrophenylphosphorylcholine is used as a substrate. Phospholipase D, calcium ion paranitrophenylphosphorylcholine + water → paranitrophenylphosphate + choline phosphate monoesterase paranitrophenylphosphate + water → paranitrophenol (yellow) + phosphoric acid

As each component of the reagent of the present invention, paranitrophenylphosphorylcholine, a substrate of phospholipase D, is 0.5 to 50 mM, preferably 1 to 10 mM.
mM, phospholipase D from 0.05 to 10 U / m
1, preferably 2 to 5 U / ml. Furthermore, glycol ether diamine tetraacetic acid, a chelating agent as an activity regulator of phospholipase D, was added to 5 to 2
00 uM, preferably 10 to 80 uM, diethylenetriaminepentaacetic acid at 1 to 50 uM, preferably 5 to 2 uM.
Use 0 uM. Phospholipase D is destabilized by this chelating agent treatment, but magnesium ion can be used as a stabilizer. Its concentration is 1
To 300 mM, preferably 10 to 100 mM. As the phosphate monoesterase, alkaline phosphatase and neutral phosphatase can be used among various enzymes of microbial and animal origin, and alkaline phosphatase derived from Escherichia coli, which has excellent stability, is preferred. Its concentration is 0.1
U / ml to 20U / ml, preferably 0.5 to 5U
/ Ml. As a buffer, keep enzyme activity stable,
Any reagent can be used as long as it can dissolve other reagents and obtain a predetermined pH, and examples thereof include various good buffers, Tris buffers, and dimethylglutarate buffers. Its pH is between 5 and 9, preferably between 6 and 7.5.
And the concentration is 5 to 500 mM, preferably 20 to 100 mM.

To determine calcium ion using the reagent of the present invention, for example, phospholipase D, alkaline phosphatase, glycol ether diaminetetraacetic acid, diethylenetriaminepentaacetic acid, paranitrophenylphosphoryl which is a substrate of phospholipase D The test solution is added to the reagent solution containing rucholine, heated to 20 to 40 ° C., and the amount of increase in absorbance of the generated paranitrophenol at an arbitrary wavelength of 380 to 450 nm may be measured.

In preparing the composition for measuring calcium ion of the present invention, the reaction reagent is divided into one or two or more, and when it is divided into two or more, those components may be appropriately combined. . The absorbance measurement can be applied not only to a spectrophotometer but also to a continuous measurement using an automatic analyzer often used in a clinical laboratory. As the test liquid,
Examples include biological fluids such as blood, for example, plasma, serum or urine, and wastewater, microbial cultures, animal and plant cultures, and biological material extracts.

[0014]

EXAMPLES Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example 1 (Reactivity of divalent cation by chelating agent treatment) 50 mM PIPES-NaOH buffer (pH
7.3) 5 mM paranitrophenylphosphorylcholine, alkaline phosphatase (1 U / ml; from Escherichia coli, Asahi Kasei T-08), 0.1% Triton X100, phospholipase D (3 U / ml; Streptomyces chromofusca) The chelating agent shown in FIG. 1 was added to a reaction solution (1 ml) composed of
And heated to 37 ° C for 2 minutes, then 10m
10 μl of a g / dl calcium or magnesium ion solution was added, and the reaction was carried out at 37 ° C. for exactly 5 minutes. Then 0.1 ml of 0.1
After adding M ethylenediaminetetraacetic acid to stop the reaction, the absorbance at a wavelength of 405 nm was measured. As shown in FIG. 1, only when glycol ether diamine tetraacetic acid was used, it did not react with magnesium ions, and the specificity of calcium ions was confirmed. However, it was found that only the treatment with glycol ether diamine tetraacetic acid reacted with ions other than magnesium ions, and the specificity was not strict (FIG. 2). It has been clarified that the combined use of glycol ether diamine tetraacetic acid and diethylene triamine pentaacetic acid almost completely eliminates the reactivity with nickel, cobalt, copper and zinc ions (FIG. 2).

[0015]

Comparative Example 1 (Specificity of magnesium ion when citrate was used as chelating agent) Sodium citrate was added as a chelating agent to a reaction solution having the composition shown in Example 1 so as to have a concentration of 10 mM. The specificity of calcium ion and magnesium ion was examined by the method described in Example 1. Also reacts with magnesium ions as shown in Table 1,
There was no specificity of the ion.

[0016]

[Table 1]

[0017]

Example 2 (Analysis by Automatic Analyzer) Calcium ions were quantified using a Hitachi automatic analyzer (model 7170). Using two types of reagents [Reagent 1; 61.1 mM P
IPES-NaOH buffer (pH 7.3), 4 U / ml
Phospholipase D (T-07 manufactured by Asahi Kasei Corporation), 5
12. 3.3 uM glycol ether diaminetetraacetic acid,
Composition of 3 uM diethylenetriaminepentaacetic acid, 33.3 mM magnesium chloride, 0.13% Triton X-100;
180 microliters, reagent 2; 5mMPIPES-
NaOH buffer (pH 7.2), composition of 16 mM paranitrophenylphosphorylcholine, 5 U / ml alkaline phosphatase, 60 microliters), and calcium ion standard solutions of various concentrations shown in FIG. C., the main wavelength 405nm,
The absorbance at a subwavelength of 660 nm was measured over time. The results are shown in FIG.

[0018]

Example 3 (Calcium ion standard curve) FIG. 4 shows a standard curve obtained from the rate of increase in absorbance of the reaction curve obtained in Example 2. The change in absorbance proportional to the calcium ion concentration was obtained as a straight line.

[0019]

Example 4 (Stabilization of Phospholipase D by Magnesium Ion) A reagent containing phospholipase D (T-07 manufactured by Asahi Kasei Corporation) having the following composition 1 to 37 was used.
After standing at 15 ° C. for 15 hours, the reagent 2 shown in Example 2 was added and the reactivity to calcium ions was examined. The results are shown in Table 2. In the composition without magnesium ion, the signal for calcium ion decreased, but the reagent containing phospholipase D could be stably stored by adding magnesium.

Composition 1: 61.1 mM PIPES-Na
OH buffer (pH 7.3), 4 U / ml phospholipase D, 53.3 uM glycol ether diaminetetraacetic acid, 13.3 uM diethylenetriaminepentaacetic acid, 0.1
A composition consisting of 3% Triton X-100. Composition 2: 61.1 mM PIPES-NaOH buffer (pH 7.3), 4 U / ml phospholipase D, 5
12. 3.3 uM glycol ether diaminetetraacetic acid,
A composition comprising 3 uM diethylenetriaminepentaacetic acid, 0.13% Triton X-100, 5 mM magnesium chloride. Composition 3: 61.1 mM PIPES-NaOH buffer (pH 7.3), 4 U / ml phospholipase D, 5
12. 3.3 uM glycol ether diaminetetraacetic acid,
A composition comprising 3 uM diethylenetriaminepentaacetic acid, 0.13% Triton X-100, and 30 mM magnesium chloride.

[0021]

[Table 2]

[0022]

According to the present invention, phospholipase D
In the presence of a chelating agent of glycol ether diamine tetraacetic acid and diethylene triamine pentaacetic acid and a magnesium ion in a calcium ion determination reagent utilizing the activation reaction of calcium ions with calcium ions, divalent cations other than calcium ions are affected. In addition, it is possible to provide a simple, highly accurate and stable reagent for determining calcium ions.

[Brief description of the drawings]

FIG. 1 is a diagram showing magnesium ion specificity when phospholipase D is treated with various chelating agents.

FIG. 2 is a graph showing the specificity of divalent cations other than magnesium ions when phospholipase D is treated with various chelating agents.

FIG. 3 is a diagram showing a change over time in a reaction when calcium ions are quantified using an automatic analyzer for clinical tests.

FIG. 4 is a diagram showing a calibration curve of calcium ions.

Claims (2)

[Claims] 1. A reagent composition for measuring calcium ion, comprising at least phospholipase D, phosphate monoesterase, a substrate for phospholipase D, glycol ether diamine tetraacetic acid, and diethylenetriaminepentaacetic acid. 2. A sample solution containing calcium ions in a calcium ion measurement reagent composition containing at least phospholipase D, phosphate monoesterase, a substrate of phospholipase D, glycol ether diamine tetraacetic acid, and diethylenetriaminepentaacetic acid. After adding
A method for measuring calcium ions, comprising measuring a phospholipase D product.