JPH0767395B2 - Method for quantifying 1,5-anhydroglucitol - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to 1,5-anhydroglucitol (hereinafter referred to as 1,5-anion) which is a diagnostic marker for diabetes.
(Hereinafter referred to as AG), which is simple and rapid and can be applied to an automatic analyzer.

[0002]

2. Description of the Related Art 1,5-AG is a compound that is present in human cerebrospinal fluid and blood, and its concentration has been reported to be significantly lowered in certain diseases, particularly in diabetes (Yasuo Akanuma, Kazuyuki Tobe: Japanese Internal Medicine). Journal of the Society, 80 1198 to 1204 19
91), which is regarded as important as a diagnostic marker for diabetes.

As a practical method for measuring 1,5-AG in a sample, saccharides other than 1,5-AG present in the sample are removed by an ion exchange column after deproteinization, and then pyranose oxidase (hereinafter referred to as PROD). Or L-
A method for quantifying hydrogen peroxide produced by oxidizing 1,5-AG with sorbose oxidase is known (see JP-A-63-185379, hereinafter, such a measuring method is referred to as a column enzyme method). .

Specimens to be measured for 1,5-AG are mainly serum or plasma of diabetic patients. Glucose concentration in the blood of the diabetic patient is higher than that of the healthy subject, and the value is about 60 to 100 mg / dl in the healthy subject, whereas it is 100 to 1000 m in the diabetic subject.
It is widely distributed in the range of g / dl. On the other hand, 1,5 in blood
-AG concentration was 1.64 to 2.68 mg / dl for healthy subjects.
On the other hand, 0.18 to 0.
It shows a remarkably low value of 21 mg / dl (Japanese clinical 47 volume 1989 extra number extensive blood / urine chemistry test immunological test first volume 439-422 Kawai). In addition, 1,5-AG
Blood levels are about 1/470 or less in diabetic patients compared to glucose. In addition, glucose and 1,
Since 5-AG has a similar chemical structure, at the present state of the art, 1,5-AG in the coexistence of glucose and 1,5-AG is used.
Selective measurement of AG is not possible, and a sample pretreatment operation for selectively removing glucose or appropriately modifying glucose is essential.

[0007] The measurement by the column enzyme method has a drawback in that the separation operation for removing deproteinized proteins and saccharides other than endogenous 1,5-AG is extremely complicated. In addition, maltose lactated Ringer's solution may be applied as an infusion to diabetic patients for the purpose of supplementation of heat source, supplementation / correction of extra-tissue fluid at the time of decrease of circulating blood volume and interstitial fluid, and correction of metabolic acidosis. Many, in this case, there is an increase in maltose in the blood of diabetic patients. Maltose in a sample from such a diabetic patient cannot be completely removed even by a separation column, and it has been reported that the 1,5-AG measurement value has a significant positive error (Clinical Chemistry 21: 43-4.
8 1992).

[0006]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The problem of the influence of endogenous maltose, which is the drawback of the conventional column enzyme method, is solved, and it is applicable to an automatic analyzer 1,5.
It is an object of the invention to provide a method for measuring AG. Another object of the present invention is to provide a method for quantifying 1,5-AG and a kit used for the method, which solves the problem of the influence of endogenous maltose and the disadvantage of complicated operation in the conventional column enzyme method. Especially.

[0007]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventor has conducted extensive studies, and as a result, 1,5-A
When quantifying G using PROD, the maltose present in the sample is reacted with α-glucosidase derived from a microorganism belonging to the genus Bacillus to convert it into glucose, and then the glucose is mixed with other saccharides present in the sample. It has been found that by selectively removing and then measuring 1,5-AG, 1,5-AG can be accurately measured without being affected by endogenous maltose.

Furthermore, the maltose present in the sample is converted to B
It is converted into glucose by the action of α-glucosidase derived from a microorganism belonging to the genus acillus , and then the glucose is mixed with other saccharides in the sample together with hexokinase (hereinafter referred to as HK) and adenosine-5′-triphosphate (hereinafter referred to as ATP). (Referred to as)) to selectively remove by phosphorylation,
After that, in the method of quantifying 1,5-AG by allowing PROD to act on 1,5-AG in the sample, the phosphorylation and the reaction of 1,5-AG with PROD are carried out particularly at pH of 7.2 to
By performing it within the range of 8.5, or as PROD, Basidiomycetus fungi N
o. By using PROD derived from 52, phosphorylation can be carried out rapidly using an excess amount of ATP, and PRO can be directly treated with ATP in the presence of an excess amount of ATP.
PROD to 1,5-AG without D being inhibited
Can be used to measure 1,5-AG, and therefore, 1,5-AG can be quantified extremely simply and quickly, and 1,5-AG applicable to an automatic analyzer can be obtained.
It was found that the quantitative method of The present invention has been completed based on these findings.

The first gist of the present invention is that 1,5-
In the method of quantifying AG using PROD, maltose present in the sample is converted into glucose by the action of α-glucosidase derived from a microorganism belonging to the genus Bacillus , and then other than 1,5-AG present in the sample. After selectively removing glucose so that 1,5-AG remains with other saccharides,
1,5-AG characterized by quantification using ROD
Is a quantitative method.

The second gist of the present invention is that 1,5-
In a method for quantifying AG using PROD, maltose present in a sample is converted into glucose by the action of α-glucosidase derived from a microorganism belonging to the genus Bacillus ; the glucose present in the sample is 1,5-
Phosphorylation in the range of pH 7.2-8.5 by HK and excess ATP along with other saccharides other than AG selectively causes 1,5-AG in the sample to remain. Removed; then PRO directly to 1,5-AG in the sample
When D is allowed to act within the range of pH 7.2 to 8.5, 1,5
A method for quantifying 1,5-AG characterized by quantifying -AG. The third gist of the present invention is 1,5-A in a sample.
In the method of quantifying G using PROD, maltose present in a sample is converted into glucose by the action of α-glucosidase derived from a microorganism belonging to the genus Bacillus ; the glucose is present in 1,5-A
Phosphorylation with HK and excess ATP along with other saccharides other than G selectively removes 1,5-AG in the sample so that it remains; then 1,5-AG in the sample as it is. To Basidiomycetous fu
ngi No. Act on 52-derived PROD 1,5
A method for quantifying 1,5-AG characterized by quantifying -AG. The fourth gist of the present invention is 1,5-A in a sample.
A kit for quantifying G, which comprises the following constituent reagents i) α-glucosidase derived from a microorganism belonging to the genus Bacillus ; ii) HK; iii) ATP; and iv) PROD; Is.

The present invention will be described in detail below. The sample in the present invention is not particularly limited as long as the concentration of 1,5-AG is to be measured, and examples thereof include serum or plasma as already mentioned. In the method for quantifying 1,5-AG of the present invention, first, maltose present in a sample is reacted with α-glucosidase to convert maltose into glucose. In order to avoid the influence of endogenous maltose originally present in the sample and apply the measurement of 1,5-AG to a general-purpose automatic analyzer, maltose in the sample is converted into glucose in a short time, and further, It is essential that glucose can be cleared in a short time. As a result of the earnest studies of the present inventor,
The α-glucosidase derived from a microorganism belonging to the genus Bacillus is known in the art, for example, Saccharomyc.
es sp. Derived α-glucosidase or Rh
izopus sp. It was found that it has a higher affinity and reactivity for maltose than the glucoamylase derived from it and that maltose in a sample can be rapidly converted to glucose. In particular, Bacillus
α-glucosidase derived from stearothermophilus is preferred. This α-glucosidase is, for example, Bacillus stearothermophili.
lus ATCC 12016 to Starch / St
aerke 1987, 39 (8), 271-273. This α-
Glucosidase is already commercially available, for example Toyobo Co.
de No. Available as AGH-211. Ba
cillus stearothermophilus
The derived α-glucosidase has the following physicochemical properties. (1) Appearance: White powder (freeze-dried) (2) Molecular weight: about 62,000 as measured by gel filtration and SDS-gel electrophoresis (3) Isoelectric point: 5.2 (4) Michaelis constant: 5.7 × 10 ^-4 M (p-nitrophenyl-α-glucose) (5) Inhibitor: heavy metal ion (Fe ²⁺ , Ca ²⁺ ) (6) Optimum pH: 6 to 7 (7) pH stability: pH 7 to Stable at 8 (8) Thermal stability: 60 ° C or less (pH 7.0, 15 minutes)
Stable at

The amount of α-glucosidase used when allowing α-glucosidase to act on maltose present in the sample is such that maltose in the sample can be completely hydrolyzed to glucose, and usually 10-200 U / ml is added. It is enough. 20 to 100 U / ml for actual measurement
Used in an amount of. The pH at which α-glucosidase acts is not particularly limited, and any pH suitable for the action of α-glucosidase may be used. Subsequent phosphorylation reaction, that is, after converting maltose to glucose by α-glucosidase, together with other sugars present in the sample,
It is preferable that the α-glucosidase is allowed to act in the same pH range as the pH range for the selective removal by phosphorylation reaction with HK and ATP, from the viewpoint of simplicity of the operation. As described below, the pH range for the phosphorylation reaction, for example, the range of pH 7.2 to 8.5, or pH 6
It is preferable that α-glucosidase is allowed to act on maltose in the sample within the range of -9. Room temperature is usually suitable.

After converting maltose present in the sample into glucose, it is selectively removed so that 1,5-AG remains together with other saccharides other than 1,5-AG present in the sample. This selective removal is sometimes referred to as the first stage reaction). Other saccharides other than 1,5-AG present in the sample mainly refer to glucose, but for example, other saccharides that are phosphorylated by HK, fructose, 5-keto-D-
Fructose, mannose, 2-deoxyglucose,
Glucosamine etc. are also targeted. 1,5-A in the sample
For the selective removal of sugars other than G, it is preferable to select a method capable of eliminating sugars such as glucose in the reaction solution without depending on any solid phase for application to an automatic analyzer. As such a method, conventionally known methods include acid decomposition of sugars using 6N hydrochloric acid, reduction treatment with sodium borohydride, conversion of glucose to gluconic acid using glucose oxidase,
There is a method of phosphorylating saccharides using HK, but among these, substances and reaction products involved in the selective removal reaction of saccharides do not have to affect the 1,5-AG quantitative reaction. The method of phosphorylating saccharides using HK is most preferable, because it allows the reaction and can complete the reaction in a short time. HK used for phosphorylating saccharides including conversion of glucose to glucose-6-phosphate is in accordance with the classification of the International Biochemical Union, EC 2.7.
It is preferable to use those classified as 1.1. In this conversion reaction, ATP and magnesium ions are used together with the enzyme. Sources of magnesium ions include organic acid salts such as fatty acid salts of magnesium, halogenated salts,
Inorganic acid salts such as sulfates, nitrates and phosphates can be used, and of these, acetates and hydrochlorides are preferable.

According to the research conducted by the present inventor, following the phosphorylation reaction in the first-step reaction, the reaction between 1,5-AG and PROD (hereinafter sometimes referred to as the second-step reaction)
By carrying out in the range of 7.2 to 8.5, an excess amount of A
Even in the presence of TP, PROD can efficiently proceed with the reaction between 1,5-AG and PROD without being inhibited by ATP. Therefore, the phosphorylation reaction in the first step reaction can be performed by using an excessive amount of ATP. pH 7.2-8.5
The phosphorylation reaction can be rapidly carried out by carrying out the reaction in the range of 1, and further, 1,5-AG and PROD are subsequently reacted in the range of pH 7.2 to 8.5 in the presence of excess ATP. This revealed that the first-step reaction and the subsequent first-step reaction, 1,5-AG and PROD, can be carried out continuously in an extremely short time. Japanese Patent Application 5-
Patent application as 022613). In addition, in the reaction between 1,5-AG and PROD in the second stage reaction, P
Especially as Basidiomycetus f as ROD
ungi No. Even when PROD derived from 52 is used, PROD does not undergo an ATP-inhibiting reaction even in the presence of an excessive amount of ATP, and therefore the first-step reaction and the subsequent second-step reaction can be carried out continuously in a short time. (It was already filed as a patent application for Japanese Patent Application No. 4-324259 for these methods).

Therefore, in the present invention, the phosphorylation reaction in the first-step reaction can be rapidly carried out by using an excessive amount of ATP. The excess amount of ATP that can be used in the phosphorylation reaction is usually 2.5 times or more ATP in molar ratio with respect to the expected glucose amount in the sample,
It is preferably 2.5 to 2500 times, more preferably 10 to
The amount is 1000 times. AT when setting up an actual measurement system
It is sufficient to adjust the amount of P to at least 5 mM. HK, AT actually used in phosphorylation reaction
Suitable amounts of P and magnesium ions are, for example, HK
Is 5 to 100 U / ml, ATP is 5 to 500 mM, and magnesium ion is 5 to 50 mM. The pH during the phosphorylation reaction is p at the time of the reaction between 1,5-AG and PROD.
Like the H range, it is preferably 7.2 to 8.5, and particularly 7.5.
〜8.0 is preferable. Also, Basid as PROD
iomycetous fungi No. When using PROD derived from 52, the pH of the phosphorylation reaction is pH
The range of 6-9 is preferable, and the range of 7.0-8.0 is particularly preferable. The temperature during the phosphorylation reaction is preferably room temperature.
The phosphorylation reaction in the first-step reaction and the above-mentioned reaction of maltose and α-glucosidase in the sample can be carried out together in the same reaction system, and the operation is carried out in the same reaction system. It is preferable in terms of simplicity.

1,5-AG is measured by a second-step reaction in which PROD is reacted with 1,5-AG remaining in a sample from which sugars other than 1,5-AG such as glucose have been selectively removed. The PROD used in the present invention is IUPA.
There is no particular limitation as long as it can be classified into EC 1.1.3.10 by the nomenclature committee of C-IUB, and various PRODs derived from strains having PROD production ability can be used. For example, Polyporus Obsass ( Polypor ) described in Japanese Patent Laid-Open No. 61-239886.
us obtusus ) ATCC 26733, a PROD derived from a strain of a microorganism belonging to the genus Polyporus, and Coriolas versicara ( Corio ) described in JP-A-58-43785.
lus versicolor IFO 4937, a PROD derived from a strain of a microorganism belonging to the genus Coriolus, and Pycnopora coccineus ( Py ) described in JP-A-62-79780.
cnoporus coccineus ) IFO492
Pikunoporasu represented by 3 (Pycnoporus)
PROD derived from a strain of a microorganism belonging to the genus, Japanese Patent Application Laid-Open No. 2-4
Basidiomycetus fung described in Japanese Patent No. 2980.
i ) No. P from 52 (Microbiology Research Institute No. 10106)
ROD, Daedaleops described in JP-A- 58-43785.
sis styracina ) IFO4910 represented by Daededaleopsis
PLOROTES OSTREATA ( Pleurotes ostretus) derived from a strain of a microorganism belonging to the genus
s ) PROD derived from a strain of a microorganism belonging to the genus Pleurotus represented by the genus Pleurotus represented by Z-64 (NRRL12507), Gloeophyllum sepiarium Z
-41 (NRRL12506), a PROD derived from a strain of a microorganism belonging to the genus Gloeophyllum , or JP-A-61-17798.
Prod derived from a strain of a microorganism belonging to the genus Irpex represented by Irpex lacteus ATCC20123 described in JP-A-6, Auricularia polytricha .
PROD derived from a strain of a microorganism belonging to the genus Auricularia represented by Z-229 (Microtechnology Research Institute No. 7119), Coprinus micaceus ATCC20
122, a PROD derived from a strain of a microorganism belonging to the genus Coprinus , or Trametes cinnab
arinus ) IFO6139, P derived from a strain of a microorganism belonging to the genus Trametes
ROD etc. are mentioned.

Among these, Polyporus obtusus ATCC2
PR derived from microorganisms belonging to the genus Polyporus such as 6733
OD, Basidiomycetaus Fungi
mycetous fung ) No. 52-derived PRO
D is preferred. In the present invention, a phosphorylation reaction using an excessive amount of ATP is carried out, and then ATP is not removed as it is, and the pH of the sample is within the range of 7.2 to 8.5.
-The enzymatic reaction of AG with PROD can be carried out. This enzymatic reaction is carried out at a pH of 7.2 to 8.5, preferably 7.5 to 8.0, so that PROD is not inhibited by ATP remaining in the sample and 1,5- PROD shows good reactivity with AG.
Or, as PROD, Basidiomyceto
us fungi No. By using PROD derived from 52, this PROD is less likely to be inhibited by ATP. Therefore, a phosphorylation reaction using an excessive amount of ATP is performed, and then the same procedure is performed continuously in the same manner as 1,5-AG.
And PROD can be carried out. In this case, the pH is preferably in the range of pH 6-9, more preferably 7.0-8.0. Also, during this oxygen reaction,
ATP in the concentration range of ˜500 mM may be present.

Hydrogen peroxide is generated by the action of PROD on 1,5-AG. Using hydrogen peroxide as a peroxidase, an enzyme classified as EC1.11.1.7 by the International Biochemical Union classification,
2'-azinobis (3-ethylbenzothiazoline-6-
Sulfonic acid), o-phenylenediamine, 5-aminosalicylic acid, 3,3 ′, 5,5′-tetramethylbenzine and 4-aminoantipyrine and N-ethyl-N-
A known substrate for peroxidase such as a combination of (2-hydroxysulfopropyl) -m-toluidine is allowed to act, and the dye produced from the substrate is measured for absorbance. The peroxidase used for measuring hydrogen peroxide is preferably horseradish peroxidase. The substrate that produces the dye used for the absorbance measurement is preferably a combination of 4-aminoantipyrine and N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine. The wavelength of the absorbance measurement range when using 4-aminoantipyrine and N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine is 500 nm to 800 nm, and in this range, two or more wavelengths are used. It can also be measured.

A suitable amount of PROD, horseradish peroxidase, 4-aminoantipyrine and N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine used in the above reaction is PROD of 5
~ 500 U / ml, horseradish peroxidase 2-20 U / ml, 4-aminoantipyrine 0.
1-10 mM, N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine is 0.1-10 mM. The effective pH range for the reaction is 7.2 to 8.5, preferably 7.5 to 8.0, or 6 to 9, preferably 7.0 to 8.0.

In the entire reaction including the first-step reaction and the second-step reaction for measuring 1,5-AG in a sample, the reaction temperature is usually room temperature, specifically 5 to 40 ° C., preferably 25. The reaction time is 2 to 60 minutes, preferably 2 to 30 minutes. When preparing the reaction solution, the whole reaction is carried out, for example, at pH 7.0 to 8.0, preferably 7.5 to 8.0.
In the reaction liquid constituting the reagent, which is a buffer solution, the pH of the reaction liquid is 7.0 to 7.0.
A phosphate buffer, a Tris-hydrochloride buffer, a HEPES buffer, or the like is used as a buffer in order to stabilize in a range of 8.0, preferably 7.5 to 8.0, and a maximum width of 6 to 9. 50 to 500 for HEPES buffer
A concentration of mM is preferred. Further, for controlling the ionic strength, an alkali metal halide salt, preferably sodium chloride, can be used. When measuring 1,5-AG by the method of the present invention, each of the above-mentioned components may be added to one solution and used, for example, the reagents used in the first-step reaction and maltose in the sample are converted into glucose. Α- which is a reagent
The solution containing glucosidase was used as the first reagent and PR
Second color development reagent for measuring OD and hydrogen peroxide
It can be used as a reagent. Alternatively, as another method, each component may be divided and used so as to be an appropriate combination. Although they may be in the form of a solution or freeze-dried, they are preferably freeze-dried when intended for long-term storage. Further, a surfactant may be added within a concentration range that does not inhibit the measurement reaction, and an appropriate amount of a stabilizer may be added when the measurement system is freeze-dried. In the present invention, the phosphorylation reaction, the enzyme reaction and the subsequent color reaction for measuring the amount of hydrogen peroxide generated can be carried out using an automatic analyzer. What can be used as an automatic analyzer in the present invention is, for example, Hitachi 7050 type, Hitachi 705 type, Hitachi 736 type.
The model is Hitachi 7150, but the model is not limited to the illustrated model, and any model similar to these may be used.

The kit used in the method for quantifying 1,5-AG of the present invention has the following constituent reagents i) α-glucosidase derived from a microorganism belonging to the genus Bacillus , as is apparent from the quantification method described above in detail. Ii) HK; iii) ATP; and iv)
A typical example is Kid containing PROD. In addition to these constituent reagents, the above-described color-developing reagent necessary for measuring hydrogen peroxide, a buffer solution necessary for pH adjustment, and the like can be added. B, one of the constituent reagents
Examples of the α-glucosidase derived from a microorganism belonging to the genus acillus include Bacillus stearthe
The α-glucosidase derived from rmophilus is preferred. As PROD, Polyporus obtu
derived from sus or Basidiomycetous
fungi No. PROD from 52 is preferred. These constituent reagents may be used in the form of a solution or in a freeze-dried state.

[0022]

EXAMPLES The present invention will now be described in more detail with reference to examples. Of course, the invention is not limited to these examples. Example 1 Maltose erasability comparison Maltose 400, 360, 320, 280, 24
0, 200, 160, 120, 80, 40, 0 mg / d
1 aqueous solution; glucose 1500, 1350, 120
0, 1050, 900, 750, 600, 450, 30
0, 150, 0 mg / dl aqueous solution; and 1,5-AG
5.0, 4.5, 4.0, 3.5, 3.0, 2.5,
The elimination reaction of maltose and glucose was examined using 2.0, 1.5, 1.0, 0.5, 0.0 mg / dl aqueous solution; as a standard sample. Furthermore, 1,5-AG quantitative reactions were compared under the following conditions.

The reagent conditions are that the first reagent does not contain the maltose hydrolase α-glucosidase, and the first reagent is the 1-A reagent; the first reagent is Saccharomyce.
s sp. Derived α-glucosidase (Toyobo Code
No. AGH-201) added with 40 KU / l is the 1-B reagent; Rhizopus s in the 1st reagent
p. Derived glucoamylase (Toyobo Code No.
AGH-111) with 40 KU / l added to the 1-
C reagent; Saccharomyces in the first reagent
sp. Derived α-glucosidase and Rhizopu
s sp. 40 KU / l of glucoamylase derived from the first reagent 1-D; Bacill in the first reagent
α derived from us stearothermophilus
-Glucosidase (Toyobo Code No. AGH-2
40 KU / l of 11) was added as the 1st-E reagent, and the other common reagent conditions and the 2nd reagent conditions had the compositions shown below. The 1-A reagent, a 1-B reagent, the 1-C reagent, common composition HEPES 200 mM NaCl 150mM magnesium acetate 10 mM N-ethyl -N- for definitive to a 1-D reagent and a 1-E reagent (2- Hydroxysulfopropyl) -m-toluidine 1 mM peroxidase 5 KU / l hexokinase 20 KU / l ATP 100 mM pH 7.5 Composition of the second reagent HEPES 200 mM sodium chloride 150 mM 4-aminoantipyrine 4 mM Polyporus obtusus- derived PROD 62.5 KU / l. 5

The reaction conditions were a standard sample volume of 7 μl, a first reagent volume of 280 μl, a second reagent volume of 70 μl, and a main wavelength of 54.
Two-point assay was carried out using a Hitachi 7150 type automatic analyzer at 6 nm and a sub wavelength of 700 nm. 1 to 3
Shows the results when using the 1-A reagent (without addition of maltose hydrolase). 1 to 4 to 6
The result when using -B reagent (added 40 KU / l of α-glucosidase derived from Saccharomyces sp.) Is shown. 7 to 9 show the results when the 1-C reagent (containing 40 KU / l of glucoamylase derived from Rhizopus sp.) Was used. 10 to 12 show the 1-D reagent ( Saccharo
myces sp. Derived α-glucosidase and Rh
izopus sp. Derived glucoamylase 40K
The results when using U / l added) are shown. 13 to 15 show the 1-E reagent ( Bacillus
α-glucosidase derived from stearothermophilus (Toyobo Code No. AGH-21
The results when (1) 40 KU / l was added) are shown.

As shown in FIG. 1, in the case where α-glucosidase which is a maltose hydrolase was not added to the first reagent, 5 m was obtained when 1,5-AG was reacted.
It shows good linearity through the origin up to g / dl, and 1500m when glucose is reacted as shown in FIG.
The absorbance was almost the same as that of the reagent blank up to g / dl. However, when maltose was reacted as shown in FIG. 3, a remarkable reaction absorbance was obtained. This means that maltose is scaled as 1,5-AG. As shown in FIGS. 4 to 6, the first reagent contains Sa.
ccharomyces sp. In the case of adding 40 KU / l of α-glucosidase derived from 1,5-A
The quantitative property of G and the scavenging property of glucose were the same as in the case where no addition was made, and almost the same result was obtained when maltose was reacted. This is Saccharomyces
sp. This indicates that maltose cannot be removed by the α-glucosidase derived from the above. As shown in FIGS. 7 to 9, Rhizopus sp. Even when 40 KU / l of glucoamylase derived from
aromyces sp. As in the case where the α-glucosidase derived from the above was added at 40 KU / l, the quantitative property of 1,5-AG and the scavenging property of glucose were the same as the case without addition, and the remarkable reaction absorbance when maltose was reacted was It was not resolved. This is Rhizopus sp.
This indicates that maltose cannot be removed even with glucoamylase derived from maltose.

As shown in FIGS. 10 to 12, Sacc
haromyces sp. A-glucosidase and Rhizopus sp. When 40 KU / l of glucoamylase derived from each was added, the same result as in the case of using each independently was obtained. This indicates that maltose cannot be removed even in the combination of these two enzymes. As shown in FIGS. 13 to 15, the Bacillus stearotherm
α-glucosidase derived from O. philus (Toyobo Co
de No. When AKU-211) was added at 40 KU / l, the quantification of 1,5-AG and the scavenging of glucose were the same as those without addition, and maltose was obtained up to a concentration of 200 mg / dl. It was possible to suppress the absorbance to almost the same level as the reagent blank. This is Bacillus stearother
α-glucosidase derived from mofilus (Toyobo C
ode No. AGH-211) has a high affinity for maltose, unlike other maltose hydrolases.
It is reactive and shows that when this enzyme is added to the first reagent, the endogenous maltose can be eliminated in a short time.

Example 2 Confirmation of Maltose Eliminating Property Maltose was added to serum of normal subjects and diabetic patients in an amount of 0 to 20.
1,5-AG was measured using the sample to which 0 mg / dl was added as a sample. The reagent conditions for the first reagent are as shown below. 1st reagent composition HEPES 200 mM sodium chloride 150 mM magnesium acetate 10 mM N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine 1 mM peroxidase 5 KU / l hexokinase 20 KU / l ATP 100 mM Bacillus stearothermophilus Derived α-glucosidase (Toyobo Code No. AGH-211) 0 or 40 or 80 KU / l pH 7.5

The second reagent was measured under the same conditions as in Example 1. That is, Bacillus stearo
The maltose erasability was confirmed by comparing the case of not adding the thermophilus- derived α-glucosidase (Toyobo Code No. AGH-211) and the case of adding 40 KU / l or 80 KU / l. The reaction conditions were as follows: sample volume 7 μl, first reagent volume 280 μl, second reagent volume 70 μl, main wavelength 546 nm, sub-wavelength 70
Two-point assay was carried out at 0 nm using Hitachi 7150 type automatic analyzer.

FIG. 16 shows that 0 to maltose was added to normal person serum.
1,5-A with 200 mg / dl added as sample
The result of having measured G is shown. Bacillus st
When α-glucosidase derived from eathermophilus (Toyobo Code No. AGH-211) was not added, almost complete error was observed when the enzyme was added at 40 KU / l or 80 KU / l, although a significant positive error due to maltose was observed. It is confirmed that the influence of maltose is avoided. FIG. 17 shows the results of measuring 1,5-AG using a sample obtained by adding 0 to 200 mg / dl of maltose to the serum of diabetic patients. Even when maltose is added to the serum of diabetic patients, it is similar to the case where maltose is added to the serum of normal subjects, as in Bacillus stearothermoph.
α-glucosidase derived from ilus (Toyobo Code
No. When AGH-211) was not added, although the significant error due to maltose was observed, the effect of maltose was almost completely avoided when the enzyme was added at 40 KU / l or 80 KU / l. It is confirmed.

Example 3 Polyporus obtu
Maltose erasability when using sus-derived PROD
Comparison with column enzyme method in the same manner as in Example 2, PRO of Polyporus obtusus derived from a sample obtained by adding 0 to 200 mg / dl of maltose to normal person serum and diabetic patient serum.
In the method of the present invention using D and the column enzyme method, 1,5-
The AG was measured. The column enzyme method was performed using Rana AG (registered trademark) of Nippon Kayaku Co., Ltd. according to the operation described in the instruction manual.

FIG. 18 shows that 0 to maltose was added to the serum of normal subjects.
1,5-A with 200 mg / dl added as sample
The result of having measured G is shown. For comparison of performance, the Bacillus ste of the present invention shown in Example 2 was used.
80% of α-glucosidase (Toyobo Code No. AGH-211) derived from Arothermophilus
The measurement results when KU / l is added are also shown. Although the column enzyme method recognizes a very significant positive error due to maltose, the method of the present invention almost completely avoids the positive error due to maltose. FIG. 19
, 0-200mg / d of maltose in serum of diabetic patients
The results obtained by measuring 1,5-AG using the sample with 1 added as a sample are shown. Also in this case, as in the case where maltose was added to normal human serum, in the column enzyme method, despite the extremely significant positive error due to maltose, in the method of the present invention, the positive error due to maltose was almost completely eliminated. I'm avoiding it.

Example 4 Basidiomycetou
s fungi No. When using PROD derived from 52
Comparison of column maltose elimination with column enzyme method Basidiomycetus fungi No.
52 except that PROD from 52 was used in the same amount in the second reagent.
According to the method and the column enzyme method of the present invention, a sample obtained by adding 0 to 200 mg / dl of maltose to normal person serum and diabetic patient serum under the same conditions as in Example 3 was used.
1,5-AG was measured. When maltose was added to normal human serum in an amount of 0 to 200 mg / dl in FIG.
1 to 0 to 200 mg / d maltose in serum of diabetic patients
The measurement result of 1,5-AG when 1 is added is shown. In the method of the present invention, the positive error due to maltose is almost completely avoided even though a significant positive error due to maltose is recognized in the column enzyme method as in the case of Example 3.

[0033]

As shown in the above examples, the measuring method of the present invention is suitable for application to an automatic analyzer and avoids the influence of endogenous maltose. That is,
The method of the present invention enables the fully automated measurement of 1,5-AG without the influence of maltose, which has been impossible in the past, and, as with many other clinical test items, a large number of specimens can be swiftly, accurately and in large quantities. I can handle it.

[Brief description of drawings]

FIG. 1 is a graph showing the quantitativeness of 1,5-AG when the 1-A reagent (without maltose hydrolase) is used.

FIG. 2 is a graph showing glucose scavenging properties when the 1-A reagent is used.

FIG. 3 is a graph showing the influence of maltose when the 1st-A reagent is used.

FIG. 4 Reagent 1-B ( Saccharomyces
sp. FIG. 4 is a graph showing the quantification property of 1,5-AG when (originating α-glucosidase added 40 KU / l) was used.

FIG. 5 is a graph showing glucose scavenging properties when the 1st-B reagent is used.

FIG. 6 is a graph showing the extent of maltose remaining when the 1-B reagent is used.

7 is a graph showing the quantification property of 1,5-AG when the 1-C reagent (containing 40 KU / l of glucoamylase derived from Rhizopus sp.) Was used.

FIG. 8 is a graph showing glucose scavenging properties when the 1-C reagent is used.

FIG. 9 is a graph showing the residual degree of maltose when the 1st-C reagent is used.

FIG. 10: Reagent 1-D ( Saccharomyces
sp. Derived α-glucosidase and Rhizopu
s sp. 3 is a graph showing the quantification property of 1,5-AG when glucoamylase derived from each is added at 40 KU / l).

FIG. 11 is a graph showing glucose scavenging properties when the 1-D reagent is used.

FIG. 12 is a graph showing the residual degree of maltose when the 1-D reagent is used.

FIG. 13: No. 1-E ( Bacillus stearo)
40 KU of thermophilus- derived α-glucosidase (Toyobo Code No. AGH-211)
FIG. 3 is a graph showing the quantitative property of 1,5-AG when (/ l added) is used.

FIG. 14 is a graph showing glucose scavenging properties when the 1st-E reagent is used.

FIG. 15 is a graph showing the erasability of maltose when the 1st-E reagent is used.

FIG. 16 shows the case of adding maltose to normal person serum,
It is a graph which shows the maltose elimination property in this invention.

FIG. 17 is a graph showing the maltose-eliminating property of the present invention when maltose was added to the serum of diabetic patients.

FIG. 18 shows the case where maltose was added to normal human serum.
It is a graph which compared the maltose elimination property in the column enzyme method and this invention.

FIG. 19 is a graph comparing the maltose elimination properties of the column enzyme method and the present invention when maltose was added to serum of diabetic patients.

FIG. 20 shows the case of adding maltose to the serum of a normal person,
It is the graph which compared the maltose elimination property in the present invention (using PROD derived from Basidiomycetus fungi No. 52) by the column enzyme method.

FIG. 21 is a graph comparing the maltose-eliminating properties of the column enzyme method and the present invention (using PROD derived from Basidiomycetaus fungi No. 52) when maltose was added to the serum of diabetic patients.

Claims (9)

[Claims] 1. A method for quantifying 1,5-anhydroglucitol in a sample using pyranose oxidase, wherein maltose present in the sample is Bacillus.
It is converted to glucose by the action of α-glucosidase derived from a microorganism belonging to the genus and then present in the sample 1,5
-With other sugars other than anhydroglucitol 1,5
A method for quantifying 1,5-anhydroglucitol in a sample using pyranose oxidase after selectively removing glucose such that anhydroglucitol remains. Determination of hydroglucitol. 2. The α-glucosidase is Bacillus
The method for quantifying 1,5-anhydroglucitol according to claim 1, which is α-glucosidase derived from stearothermophilus . 3. A method for quantifying 1,5-anhydroglucitol in a sample using pyranose oxidase, wherein maltose present in the sample is converted into glucose by the action of α-glucosidase derived from a microorganism belonging to the genus Bacillus. Converted; the glucose, along with other sugars other than 1,5-anhydroglucitol present in the sample, with hexokinase and excess adenosine-5'-triphosphate to a pH of 7.2-8. Phosphorylation within the range of 5 selectively removes 1,5-anhydroglucitol in the sample so that it remains; then 1,5-anhydroglucitol in the sample is directly converted to pyranose. 1,5-anhydroglucitol is quantified by causing oxidase to act within a pH range of 7.2 to 8.5, 1,5- Determination of anhydroglucitol. 4. The pyranose oxidase is Polyp
The method for quantifying 1,5-anhydroglucitol according to claim 3, which is a pyranose oxidase derived from Orus obtusus . 5. A method for quantifying 1,5-anhydroglucitol in a sample using pyranose oxidase, wherein maltose present in the sample is converted into glucose by the action of α-glucosidase derived from a microorganism belonging to the genus Bacillus. In the sample by phosphorylating the glucose with hexokinase and excess adenosine-5'-triphosphate along with other sugars other than 1,5-anhydroglucitol present in the sample. 1,5-
Anhydroglucitol was selectively removed so as to remain; and then 1,5-anhydroglucitol in the sample was directly added to Basidiomycetous fungi.
No. A method for quantifying 1,5-anhydroglucitol, which comprises quantifying 1,5-anhydroglucitol by allowing 52-derived pyranose oxidase to act. 6. The method for quantifying 1,5-anhydroglucitol from the amount of hydrogen peroxide produced by reacting pyranose oxidase with 1,5-anhydroglucitol in a sample. 1,5 according to any one of 5
Determination of anhydroglucitol. 7. A kit used for quantifying 1,5-anhydroglucitol in a sample, which comprises the following constituent reagents: i) α-glucosidase derived from a microorganism belonging to the genus Bacillus ; ii) hexokinase; iii) A kit for quantifying 1,5-anhydroglucitol, which comprises: adenosine-5'-triphosphate; and iv) pyranose oxidase. 8. The pyranose oxidase is Polyp
orus obtusus- derived pyranose oxidase or Basidiomycetous fungi
No. The kit for quantifying 1,5-anhydroglucitol according to claim 7, which is 52-derived pyranose oxidase. 9. The 1,5-an according to claim 7, which contains a chromogenic reagent necessary for quantifying the amount of hydrogen peroxide produced during the quantification of 1,5-anhydroglucitol. A kit for quantifying hydroglucitol.