JP2990753B2 - Composition for chlorine ion measurement - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a composition for measuring chloride ions. Measurement of chloride ions in body fluids provides useful information on abnormal transport, absorption and elimination of various electrolytes in a living body, and has high clinical significance.

(Prior art) Conventionally, the measurement of chloride ions in body fluids includes a method for measuring chloride ion concentration as an electric signal, such as a coulometric titration method using a chloride meter and an electrode method using an ion selective electrode, and mercury thiocyanate. A colorimetric method using iron nitrate has been generally used. However, the former coulometric titration method,
The electrode method requires a dedicated device, and requires care in holding and managing the device, and has a problem that the efficiency of sample analysis is low. In the latter colorimetric method, cyan
There is a drawback that a special treatment is required because a waste liquid containing mercury is generated. In addition, recently, a method for measuring chloride ion using an enzymatic method, which comprises an inactive α-amylase and a reagent for measuring α-amylase (Japanese Patent Application Laid-Open
63-126497) has come to be used. But,
This method is a method in which a calcium complex is added to a maltooligosaccharide derivative for measurement, and has a problem that the reagent blank rises significantly and the accuracy of the measured value is poor. Further, there is also a problem that the measurement range is narrow, and a correct measurement value is not given in a high concentration range higher than a normal value.

(Problems to be Solved by the Invention) The present invention is to solve the above-mentioned conventional problems, and it is an object of the present invention to eliminate the necessity of holding and managing special equipment, to improve the analysis efficiency, and to special waste liquid. No processing is required,
An object of the present invention is to provide a simple chloride ion measuring reagent which can suppress a rise in a reagent blank, has excellent measurement accuracy, has good linearity, and provides an accurate measurement value even for a high-value sample.

(Means for Solving the Problems) The present inventors have conducted intensive studies in order to achieve the above object, and have reached the present invention.

That is, the present invention provides a maltooligosaccharide derivative having a modified or unmodified non-reducing end and a modified reducing end, a metal chelator, an α-amylase and a tracking enzyme, for chloride ion measurement. A composition.

The maltooligosaccharide derivative used in the present invention refers to a maltooligosaccharide derivative having a modified non-reducing end represented by the following general formula (I) and a modified reducing end, and an unmodified non-modified non-reducing end represented by the following general formula (II). Maltooligosaccharide derivatives having a reducing end and a modified reducing end.

General formula (I) (Wherein at least one of R ¹ and R ² has 1 to 1 carbon atoms)
Represents 6 alkyl groups, substituted alkyl groups, phenyl groups or substituted phenyl groups, the remaining groups represent hydrogen atoms, or R ¹ and R ² together form a methylene bridging group Often, the hydrogen atom of this bridging group has 1 to 1 carbon atoms.
It may be substituted by an alkyl group having five, a substituted alkyl group, a phenyl group, or a substituted phenyl group. R ³ represents a phenyl group or a substituted phenyl group. n is 1
Shows an integer of ~ 8. ) General formula (II) (In the formula, R ³ represents a phenyl group or a substituted phenyl group.
n shows the integer of 1-8. The maltooligosaccharides of the maltooligosaccharide derivatives used in the present invention include maltotriose, maltotetraose,
Maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose, maltodecaose and the like. Particularly, maltotriose, maltotetraose and maltopentaose are preferred.

4- of the non-reducing terminal glucose of the maltooligosaccharide
The modifying group for modifying the hydroxyl group at the 1-position and / or 6-position may be a group having 1 to 1 carbon atoms such as methyl, ethyl and propyl.
6 alkyl groups, hydroxymethyl, hydroxyethyl, sulfomethyl, carboxymethyl, sulfoethyl,
Substituted alkyl groups such as carboxyethyl, 2-ketopropyl, 2-ketobutyl, 2-ketopentyl, 3-ketopentyl, 4-ketopentyl, and benzyl; a phenyl group;
Substituted phenyl groups such as 4-methylphenyl, 4-hydroxyphenyl, 4-carboxyphenyl, 4-sulfophenyl, 2-ketopropylidene, 2-ketobutylidene,
3-ketobutylidene, 2-ketopopentyliden, 3-ketopopentyliden, 4-ketopopentyliden, benzylidene,
Methylidene, ethylidene, isopropylidene, cyclohexylidene, methylsulfinylethylidene, ethylsulfinylethylidene, methanesulfonylethylidene,
There are methylene bridging groups such as ethanesulfonylethylidene.

In particular, the hydroxyl groups at the 4-position and 6-position of the non-reducing terminal glucose are preferably bonded by a methylene bridge group, and the hydrogen atom of the methylene bridge group is a polar group such as a carbonyl group, a sulfinyl group, and a sulfonyl group. Is preferably substituted with an alkyl group having the formula:

The modifying group for modifying the hydroxyl group at the 1-position of the reducing terminal glucose of the maltooligosaccharide includes a phenyl group and a substituted phenyl group, and the substituted phenyl group has a substituent such as halogen, nitro, or hydroxy. Such substituted phenyl groups include 4-nitrophenyl, 2-chloro-4-nitrophenyl, 2,6-dichloro-4-nitrophenyl, 2-fluoro-4-nitrophenyl, 2,6-
Difluoro-4-nitrophenyl, 2-bromo-4-nitrophenyl, 2,6-dibromo-4-nitrophenyl,
There are 2-nitrophenyl, 2-hydroxy-4-nitrophenyl, 3-hydroxy-4-nitrophenyl group and the like. Particularly, a nitro group and / or a phenyl group having a halogen atom are preferable. These groups are bonded to the hydroxyl group at the 1-position of the reducing terminal glucose in α-type or β-type.

Examples of the maltooligosaccharide derivative used in the present invention include 3-ketobutylidene-2-chloro-4-nitrophenyl-D-maltotrioside, 3-ketobutylidene-
2-chloro-4-nitrophenyl-D-maltotetraoside, 3-ketobutylidene-2-chloro-4-nitrophenyl-D-maltopentaoside, ethylidene-4
-Nitrophenyl-D-maltotrioside, ethylidene-4-nitrophenyl-D-maltotetraoside,
Ethylidene-4-nitrophenyl-D-maltopentaoside, benzylidene-4-nitrophenyl-D-maltotrioside, benzylidene-4-nitrophenyl-
D-maltotetraoside, benzylidene-4-nitrophenyl-D-maltopentaoside, benzyl-4-
Nitrophenyl-D-maltotrioside, benzyl-
4-nitrophenyl-D-maltotetraoside, benzyl-4-nitrophenyl-D-maltopentaoside, 2-chloro-4-nitrophenyl-D-maltotrioside, 2-chloro-4-nitrophenyl- D-maltotetraoside, 2-chloro-4-nitrophenyl-
D-maltopentaoside, 4-nitrophenyl-D-
A reducing end-modifying group such as maltotrioside, 4-nitrophenyl-D-maltotetraoside, 4-nitrophenyl-D-maltopentaoside or the like is bonded in α-type or β-type, or And a maltooligosaccharide derivative which is a mixture of β-type bonds.

As the metal chelating agent used in the present invention, ethylenediaminetetraacetic acid, ethylenediamine-N, N, N ', N'-
Tetraacetic acid, trans-1,2-cyclohexanediamine-N,
N, N ', N'-tetraacetic acid, dihydroxyethylglycine, diaminopropanoltetraacetic acid, diethylenetriaminepentaacetic acid, ethylenediaminediacetic acid, ethylenediaminedipropionate hydrochloride, hydroxyethylenediaminetriacetic acid,
Ethylenediaminetetrakis (methylenephosphonic acid),
Glycol ether diamine tetraacetic acid, hexamethylene diamine-N, N, N'-N ',-tetraacetic acid, hydroxyethyliminodiacetic acid, iminodiacetic acid, diaminopropanetetraacetic acid, nitrilotriacetic acid, nitrilotripropionic acid, nitrilotris (Methylene phosphonic acid), triethylenetetramine hexaacetic acid, 1,2-bis (ortho-aminophenoxy)
Ethane-N, N, N ', N'-tetraacetic acid and their monovalent metal salts. These can be used alone or in combination of two or more.

The α-amylase used in the present invention is derived from human pancreas,
It is derived from human saliva, from pigs, from cattle, from microorganisms, and the like.

When a maltooligosaccharide derivative having a modified non-reducing end is used, all α-amylases derived from humans, other animals and microorganisms are applicable. When a maltooligosaccharide derivative having an unmodified non-reducing end is used, α-amylase derived from porcine pancreas is preferred.

As the following enzyme, at least one enzyme selected from the group consisting of α-glucosidase, β-glucosidase and glucoamylase is used.

When only β-glucosidase is used, a maltooligosaccharide in which a modifying group is bonded to the 1-position hydroxyl group of the reducing terminal glucose of maltooligosaccharide in β form, or a mixture of α form and β form bond A sugar derivative is used.

The origin of the α-glucosidase used in the present invention is not particularly limited. Α-Glucosidase obtained from animals, plants, microorganisms and the like can be used.

The origin of β-glucosidase and glucoamylase is not particularly limited. For example, β obtained from almonds
-Glucosidase or glucoamylase obtained from Rhizopus delemer can be suitably used.

Phosphate buffer solution in the composition for measuring chloride ion of the present invention,
Any buffer such as citrate buffer, borate buffer or Good buffer can be used. However, those containing chlorides such as hydrochloric acid and sodium chloride are not suitable. Buffer pH is 7.0
Near is preferred.

The composition for measuring chloride ion of the present invention includes a maltooligosaccharide derivative 0.1 to 20 mM, a chelating agent 0.01 to 200 mM, α-amylase 0.05 to 50 u / ml, and α-glucosidase 0 to 200 u / m.
1, β-glucosidase 0-50 u / ml, glucoamylase 0-50 u / ml. As a follower enzyme, at least
Requires 0.01u / ml.

The composition for measuring chloride ion of the present invention contains a surfactant, a preservative, a stabilizer and the like as necessary.

In order to measure the chloride ion in the sample, a reagent containing a maltooligosaccharide derivative, a metal chelating agent, α-amylase and a tracking enzyme is added to the sample, and the resulting phenol or substituted phenol is measured optically. Further, the reagents may be divided into two or more and appropriately combined. For example, a metal chelating agent, α-amylase, a first reagent containing a tracking enzyme and a second reagent containing a metal chelating agent and a maltooligosaccharide derivative, and after adding the first reagent to the sample, adding the second reagent and reacting, The resulting phenol or substituted phenol is measured optically.

The composition for chlorine ion measurement of the present invention has a high analytical efficiency,
No special waste liquid treatment is required, the rise of the reagent blank can be suppressed, the measurement accuracy is excellent, the linearity is good, and the measurement can be performed accurately and easily up to a high concentration range. The reagent according to the present invention is applicable to continuous measurement using an automatic analyzer.

(Examples) Hereinafter, the present invention will be described with reference to examples.

Example 1 A reagent blank was raised by the following method using the following reagent,
The concentrations of chloride ions in three types of serum were measured.

1. Reagent Enzyme reagent (referred to as R1) 0.15 M phosphate buffer (pH 7.0) 38 mM EDTA 20 U / ml α-amylase (porcine pancreas) 110 U / ml α-glucosidase 3 U / ml β-glucosidase Maltooligosaccharide derivative reagent (Referred to as R2) 0.15M phosphate buffer (pH 7.0) 38mM EDTA 3.7mM Maltooligosaccharide derivative (described in Table 1) Similarly, a test solution was prepared by adding 0.75mM Ca-EDTA for both R1 and R2. Let it be Comparative Example 1.

2.Measurement method After adding 2ml of the above R1 test solution to 25μ test solution and leaving it at 37 ° C for 5 minutes, add 500μR R2 test solution and react at 37 ° C.
The absorbance was measured at a wavelength of 400 nm to determine the amount of change in absorbance per minute. The concentration of serum was calculated using 70 mM KCl as a standard solution.

As is evident from the results, all of the present invention obtained good measured values with a lower reagent blank rise as compared with Comparative Example 1. 3KB-G5-β-C with specially modified non-reducing ends
It was good when NP, E-G7-α-PNP was used.

Example 2 Using the same reagents and method as in Example 1 above, and using KC1 solutions having different concentrations as test liquids, the linearity was examined and the measurable range of each was determined. The results are shown in FIG. 1 and FIG. From Figure 2, 3KB-G with modified non-reducing end
It is evident that the use of 5-β-CNP shows good linearity, especially at high concentrations, and is an excellent composition for measuring chloride ions with a wide measurement range. Also, the first
The figure shows that even when G5-β-CNP whose non-reducing terminal is not modified is used, good linearity is exhibited in the chloride ion concentration range of 50 to 140 mM.

Comparative Example 2 FIG. 3 shows the result of examining the linearity of the same reagent as in Comparative Example 1 above, using 3KB-G5-β-CNP as a maltooligosaccharide derivative, in the same measurement method as in Example 2 above. Show. When the reagent contains a calcium complex, the linearity is poor and it can be seen that accurate measurement cannot be performed.

Example 3 A reagent blank was raised by the following method using the following reagent,
The concentrations of chloride ions in three types of serum were measured.

1. Reagent Enzyme reagent (referred to as R1) 0.15M phosphate buffer (pH 7.0) 38mM EDTA 20U / ml α-amylase (porcine pancreas) 1U / ml α-glucoamylase 3U / ml β-glucosidase maltooligosaccharide derivative Test solution (referred to as R2) 0.15M phosphate buffer (pH 7.0) 38mM EDTA 3.7mM maltooligosaccharide derivative (described in Table 3) Similarly, a test solution was prepared in which both R1 and R2 were added with 0.75mM Ca-EDTA. And Comparative Example 3. 2. Measurement method Measurement was performed in the same manner as in Example 1.

Table 4 shows the results of Example 3 and Comparative Example 3. It is clear from the results of Example 3 that chloride ion can be measured even when glucoamylase is used as the following enzyme. Particularly, when 3KB-G5-β-CNP having a non-reducing terminal modified is used, a reagent blank is used. The rise of the value was small and good results were obtained.
Also, when G7-β-CNP and G7-α-PNP were used, the increase in the reagent blank was small and good as compared with Comparative Example 3.

Example 4 A reagent blank was raised by the following method using the following reagent,
The concentrations of chloride ions in three types of serum were measured.

1. Reagent Enzyme reagent (referred to as R1) 0.15 M phosphate buffer (pH 7.0) 38 mM EDTA 20 U / ml α-amylase (described in Table 5) 110 U / ml α-glucosidase 3 U / ml β-glucosidase maltooligosaccharide Derivative reagent solution (referred to as R2) 0.15 M phosphate buffer (pH 7.0) 38 mM EDTA 3.7 mM maltooligosaccharide derivative (described in Table 3) 2. Measurement method Measurement was performed in the same manner as in Example 1.

Table 6 shows the measurement results. Even if the origin of the α-amylase used was different, measurement of chloride ion was possible.
When a maltooligosaccharide having a non-reducing end modified is used,
There was no difference in the reagent blank due to the difference in the origin of α-amylase, and the blank change was small.

Example 5 A reagent blank was raised by the following method using the following reagent,
The concentrations of chloride ions in three types of serum were measured.

1. Reagent A Enzyme reagent (referred to as R1) 0.15M phosphate buffer (pH 7.0) 100mM EDTA 21.5U / ml α-amylase (derived from porcine pancreas) 5U / ml β-glucosidase maltooligosaccharide derivative reagent (referred to as R2) 0.15 M phosphate buffer (pH 7.0) 100 mM EDTA 3.5 mM Maltooligosaccharide derivative (listed in Table 7) Reagent B Enzyme TS (referred to as R1) 0.15 M phosphate buffer (pH 7.0) 100 mM EDTA 21.5 U / ml α-amylase (derived from pig pancreas) 110U / ml α-glucosidase 5U / ml β-glucosidase Maltooligosaccharide derivative test solution (referred to as R2) 0.15M phosphate buffer (pH 7.0) 100mM EDTA 3.5mM G5-β- CNP 2. Measuring method Add 2 ml of the above R1 test solution to 25 µ of the test solution, leave at 37 ° C for 5 minutes, add 500 µ of the R2 test solution, and react at 37 ° C.
The absorbance was measured at a wavelength of 400 nm to determine the amount of change in absorbance per minute. The concentration of serum was calculated using 70 mM KCl as a standard solution. The results are shown in Table 8.

As is evident from these results, for maltooligosaccharides in which a modifying group is β-linked to the reducing end, when only β-glucosidase is used as a follow-up enzyme, all good results are obtained, and in particular, as a follow-up enzyme, α-glucosidase,
The blank rise was smaller and better than when β-glucosidase was included.

Comparative Example 4 The following reagents were used to increase the serum concentration of three kinds of chloride ions in the same manner as in Example 5 using the following reagents.

Enzyme reagent (referred to as R1) 0.15M phosphate buffer (pH 7.0) 100mM EDTA 1mM Ca-EDTA 21.5U / ml α-amylase (derived from porcine pancreas) 5U / ml β-glucosidase maltooligosaccharide derivative reagent (referred to as R2) ) 0.15 M phosphate buffer (pH 7.0) 100 mM EDTA 1 mM Ca-EDTA 3.5 mM G5-β-CNP The results are shown in Table 9.

It can also be seen that when the reagent contains only β-glucosidase as the following enzyme, the addition of the calcium complex significantly increases the blank rise.

Example 6 The same reagent as the reagent A of Example 5 described above, but using G5-β-CNP as a maltooligosaccharide derivative, using the same method, and using a KCl solution having a different concentration as a test liquid, to obtain a dilution linearity. Was examined. The result is shown in FIG.

It is clear that the composition according to the invention has good linearity and can give accurate measurements even for high-value analytes.

(Effect of the Invention) According to the present invention, by using a maltooligosaccharide derivative having a modified or unmodified non-reducing end and a modified reducing end, the rise of the reagent blank can be suppressed with high accuracy and easily to a high concentration. Measurement of chloride ion can be performed.

[Brief description of the drawings]

FIG. 1 shows that 2-chloro-4- as a maltooligosaccharide derivative
Nitrophenyl-β-D-maltopentaoside (G5-
4 shows a calibration curve of a KCl solution when (β-CNP) is used. FIG. 2 shows KC when 3-ketobutylidene 2-chloro-4-nitrophenyl-β-D-maltopentaoside (3KB-G5-β-CNP) was used as a maltooligosaccharide derivative.
1 shows a calibration curve of the solution. FIG. 3 shows a calibration curve of a KCl solution when 3KB-β-CNP is used by adding a calcium complex. FIG. 4 shows the dilution linearity of the KCl solution when only β-glucosidase was used as the following enzyme.

Continuation of the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C12Q 1/40 C12Q 1/34

Claims (5)

(57) [Claims] (1) It comprises a maltooligosaccharide derivative having a modified or unmodified non-reducing end and a modified reducing end, a metal chelating agent, α-amylase and a following enzyme, and does not contain a calcium complex. Composition for measuring chloride ions. 2. The composition according to claim 1, wherein the tracking enzyme is at least one enzyme selected from the group consisting of α-glucosidase, β-glucosidase and glucoamylase. 3. The composition for measuring chloride ion according to claim 1, wherein the following enzyme is β-glucosidase only. 4. A maltooligosaccharide derivative having a modified non-reducing end and a modified reducing end is represented by the following general formula (I): (Wherein at least one of R ¹ and R ² has 1 to 1 carbon atoms)
Represents six alkyl groups, substituted alkyl groups, phenyl groups, or substituted phenyl groups, and the remaining groups represent hydrogen atoms, or R ¹ and R ² together form a methylene bridging group. Often, the hydrogen atoms of this bridging group may be substituted by alkyl, substituted alkyl, phenyl, or substituted phenyl groups having from 1 to 5 carbon atoms. R ³ represents a phenyl group or a substituted phenyl group having a substituent selected from the group consisting of halogen, nitro and hydroxy. n shows the integer of 1-8. The composition for measuring chloride ion according to claim 1, which is a compound represented by the formula: 5. A maltooligosaccharide derivative having an unmodified non-reducing end and a modified reducing end is represented by the following general formula (II): (Wherein, R ³ represents a phenyl group or a phenyl group having a substituent selected from the group consisting of halogen, nitro and hydroxy. N represents an integer of 1 to 8). Item (1). The composition for measuring a chlorine ion according to Item (1).