JP2671104B2 - Enzyme reagent for quantifying 1,5-anhydroglucitol - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to the quantification of 1,5-anhydroglucitol (hereinafter referred to as 1,5-AG), which is expected as a diagnostic marker for diabetes. It relates to reagents. [0002] 1,5-AG is a compound which is present in human cerebrospinal fluid and plasma, and has been reported to decrease its plasma level in certain diseases, particularly in diabetes. This method for quantifying 1,5-AG has been conventionally mainly performed by gas chromatography. (Diabetes 25
[0003] However, the conventional method requires pretreatment of a test solution and labeling of 1,5-AG. In addition, the maintenance and management of analytical equipment requires sophisticated techniques and is complicated, and gas chromatography requires a long time for analysis, making it difficult to measure many test liquids. There was a problem with the quantification method. Therefore, the present inventors studied a simple and easy measurement method capable of measuring a large number of cases, and completed the present invention. [0004] The present invention, for example, in combination with a sugar removing agent, a reagent for detecting hydrogen peroxide, 1,5-AG
1,5 containing pyranose oxidase or L-sorbose oxidase, which can be a reagent kit for quantification
-It relates to an enzyme reagent for quantifying AG. Here, as a sugar removing agent, boric acid,
Examples thereof include boric acid-binding resin, strongly basic anion exchange resin, hydrochloric acid, sodium borohydride, glucose oxidase, hexokinase, anion exchange resin, cation exchange resin, etc. However, boric acid binding resin is easy to operate. , A combination of an anion exchange resin and a cation exchange resin, and a combination of a strongly basic anion exchange resin and a cation exchange resin are preferred, and usually a small disposable column with boric acid binding resin in the kit. Or a highly basic anion exchange resin packed in the uppermost layer. Next, a method for removing sugars from a specimen will be described. When a sample is treated with a strongly basic anion exchange resin or boric acid, 1,5-AG is not removed at all, and saccharides are selectively removed to obtain a sample containing 1,5-AG.
The method using a strongly basic anion exchange resin is one in which a sample is slowly passed through the OH form of the strongly basic resin to adsorb and remove saccharides. Preferred strongly basic resins are resins having a quaternary ammonium salt as an exchange group, such as anion exchange resins having a strongly basic trimethylamino group (I type) or a hydroxyethyldimethylamino group (II type). Further, in this method, since the processing liquid is affected by the passing speed of the resin, it is preferable that the particle size of the resin is fine (200 to 400 mesh) and the resin is passed slowly. Further, in order to neutralize the treatment solution, the treatment solution may be further treated with a cation exchange resin. The cation exchange resin is an H-type of various anionic resins. As anionic resin, from strongly acidic cation exchange resin to weakly acidic cation exchange resin,
Includes all types of cation exchange resins. Particularly preferred is H-form of a strongly acidic cation exchange resin having a sulfonic acid group. In the method using boric acid, the specimen may be treated with boric acid itself, but it is preferable to use a resin to which boric acid is bound. Examples of the resin to which boric acid is bound include a boric acid covalent bond type resin and a boric acid type anion exchange resin. [0008] The boric acid type anion exchange resin is a borate type of various cationic resins. The cationic resin may be a strong basic anion exchange resin to a weak basic anion. Includes all types of anion exchange resins up to the exchange resin. Particularly preferably, a borate form of an anion exchange resin having a strongly basic trimethylamino group (type I), a strongly basic hydroxyethyldimethylamino group (I
(I-type) and a borate type of anion-exchange resin. When this boric acid-treated solution is used as a sample, an anion-exchange resin or an anion-exchange resin and a cation-exchange resin are used to remove a complex of boric acid and a saccharide that cannot be completely removed, which is present in the solution. It is better to process. The anion exchange resin is an OH form or a weak acid salt form of various cationic resins. Examples of the cationic resin include a strong basic anion exchange resin and a weak basic anion. It includes all types of anion exchange resins, up to the exchange resin. Organic acids such as carbonic acid, formic acid and acetic acid are preferable as the weak acids constituting the weak acid salt form of these cationic resins. Particularly preferred cationic resins include anion exchange resins having a strongly basic trimethylamino group (type I) or a hydroxyethyldimethylamino group (type II). Examples of the cation exchange resin include those described above. To carry out the boric acid treatment method for removing saccharides, for example, the following method may be used. When boric acid itself is used, an aqueous solution of boric acid is used as a boric acid in a test solution such as plasma, and the boric acid is added to such an extent that boric acid is not leaked in an anion exchange resin treatment in the next step, followed by stirring. Next, the treatment liquid may be passed through a column filled with an anion exchange resin or an anion exchange resin and a cation exchange resin. When a resin to which boric acid is bound is used, the resin, the anion exchange resin and the cation exchange resin are packed in a column such that the boric acid-bound resin is at the top, and then the column is put on the column. What is necessary is just to let a sample pass. [0011] In addition to the above-mentioned methods, methods for selectively removing saccharides from a sample include a method utilizing the extreme chemical stability of 1,5-AG, and a method other than 1,5-AG. There is a method of modifying glucose, which is a major contaminant substrate, using an enzyme as a catalyst. As a method utilizing the chemical stability of 1,5-AG, heating in the presence of 6N hydrochloric acid and
A method of acid-decomposing saccharides other than G and recovering 1,5-AG remaining in the decomposition product, and treating with a reducing agent such as sodium borohydride to obtain glucose other than 1,5-AG And the like, and reducing the saccharide having a carbonyl group or formyl group to a compound which cannot react with the enzyme for quantifying 1,5-AG. On the other hand, in the method of chemically modifying an enzyme as a catalyst, an enzyme that converts glucose to gluconic acid, for example, a method using glucose oxidase (EC1,1,3,4), or converting glucose to glucose-6-phosphate is used. There is a method using an enzyme, for example, hexokinase (EC2, 7, 1, 1). The compounds modified by these enzymes are compounds that cannot react with pyranose oxidase and L-sorbose oxidase, which are 1,5-AG quantification enzymes, and remain without any change by the enzyme treatment. 1,5-AG may be measured by the following method. Examples of the reagent for detecting hydrogen peroxide include a combination of peroxidase or a peroxidase-like active substance and a chromogenic substrate or a chromogenic agent and a coupler, a combination of peroxidase and a luminescent substrate, a combination of peroxidase and a luminescent substrate, and a combination of ferricyan ion and a luminescent substrate. . The method for detecting hydrogen peroxide used in the present invention may be any method that can be detected with high sensitivity,
Numerous methods are available. The most commonly used of these methods is to oxidize various HRP substrates with hydrogen peroxide using horseradish peroxidase (HRP) as a catalytic enzyme. The light substance and the chemiluminescence may be measured by absorbance measurement, fluorescence measurement and luminescence measurement, respectively. As an HRP substrate for producing a dye, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABT
S), o-phenylenediamine (OPD), 5-aminosalicylic acid (5-AS), 3,3 ', 5,5'-tetramethylbenzidine (TMB) and the like. Substrates for HRP that generate a fluorescent substance include p-hydroxyphenylacetic acid and 3- (p-hydroxyphenyl) propionic acid (HP
PA). Luminol and isoluminol are known as chemiluminescent HRP substrates. Specifically, for example, as follows: ## STR1 ## Sodium phosphate buffer solution (1/15 M, pH 5.6)
3ml, 4mM 2,2'-azinobis [3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) and 12unit / ml
Coloring solution containing horseradish peroxidase
5 ml, 0.1 unit of 25 unit / ml pyranose oxidase or L-sorbose oxidase solution and 40 μg / ml of 1,
0.1 ml of the 5-AG solution is placed in a container and reacted at 37 ° C. for 30 minutes. The reaction is stopped under ice cooling, and the absorbance at 405 nm is measured. A calibration curve is prepared using a 1,5-AG solution having a known concentration, and the concentration of 1,5-AG is calculated from the absorbance of the sample. Several methods for chemiluminescent detection without HRP are also known. For example, a method of emitting luminol with hydrogen peroxide in the presence of ferricyan ions, a method of emitting lucigenin with hydrogen peroxide in the presence of metal ions, an allyl oxalic acid such as bis (2,4,6-trichlorophenyl) oxalate There is known a method in which an ester compound is reacted with hydrogen peroxide in the presence of a fluorescent substance to excite the fluorescent substance with the decomposition energy of the oxalate to emit light. Further, as a method of directly detecting hydrogen peroxide, a hydrogen peroxide electrode may be used. The enzyme reagent for quantifying 1,5-AG of the present invention is a reagent containing pyranose oxidase (PROD) or L-sorbose oxidase. It was first revealed by the present invention that pyranose oxidase or L-sorbose oxidase can be used for quantifying 1,5-AG. Pyranose oxidase and L-sorbose oxidase are not particularly limited as long as they can be classified into EC11310 and EC11311 by the IUPAC-IUB Nominal Commission, respectively. Examples of pyranose oxidase include polyporous oxidase. Sus (Polyporus obtu
sus) ATCC26733 produced, and L
Examples of sorbose oxidase include those produced by Trametes anguines IFO4923. In the reagent of the present invention, these enzymes are usually used after being dissolved in a buffer solution, but they may be used after being immobilized by a conventional method such as microcapsules, those covalently bound to or adsorbed on resins and polysaccharides. .
The amount of enzyme used varies depending on the amount of the sample and the like, but in order to make the reaction time by the enzyme a suitable short time, it may be 1 unit or more, and preferably about 1 to 5 units. It goes without saying that the higher the specific activity of the enzyme used is, the better the reaction is, but it is not always required to have the highest purity. The method for collecting the enzyme used in the present invention is, for example, Bioch-im. Biophys. Acta 167, 493-500 (1968) for the former.
For the latter, see The Journal of Biochemistry 62.
(2) 223-229 (1967). The enzyme reagents for quantifying 1,5-AG and the reagents for detection, which are the components of the kit, may be mixed to form a single reagent, and if there are components that interfere with each other, The components may be divided into suitable combinations. These may be prepared as a solution or a powdery reagent, or may be contained in a suitable support such as filter paper or film to prepare a test paper or a film for analysis. In addition to the above combination reagents, a standard reagent containing a deproteinizing agent such as perchloric acid or a fixed amount of 1,5-AG may be attached to the analytical reagent kit. The amount of the removing agent for substrates other than 1,5-AG in the kit is, for example, when using a strongly basic anion exchange resin. Strongly basic anion exchange resin (OH form) 0.1 to 1.0 ml Cation exchange resin 0.01 The amount is preferably about 0.5 ml, and may be appropriately adjusted so that the total amount of the resin is 0.2 to 1.5 ml. When a boric acid-binding resin is used, boric acid-binding resin is 0.1 to 0.5 ml, anion exchange resin is 0.1 to 1.0 ml, cation exchange resin is about 0.01 to 0.5 ml, and it is appropriately adjusted so that the total amount of the resin is 0.5 to 2 ml. Good. The amount of enzyme in the enzyme reagent for quantifying 1,5-AG in the kit varies depending on the number of measurable samples such as 100 samples and 300 samples, but is about 0.5 to 20 units per sample,
As for the detection reagent, when HRP and ABTS are used as hydrogen peroxide detection reagents, HRP is added to the kit at 10 to 200 m unit and ABTS at 1 to 20 μmol per sample. In addition, it is preferable to incorporate 5 to 20 μl of perchloric acid as 100% as a sample as a protein removing agent and 100 to 1000 μg of 1,5-AG as a sample for preparing a calibration curve in a kit. The sample for quantifying 1,5-AG is
There is no particular limitation as long as 1,5-AG is to be quantified, and examples thereof include cerebrospinal fluid, plasma, serum, and urine. Since saccharides such as glucose that can react with pyranose oxidase or L-sorbose oxidase are present in these samples, a sample from which the saccharides have been removed is used. In order to quantify 1,5-AG using the above-mentioned kit, an electron acceptor and 1,5-AG quantifying enzyme are added to a sample obtained by treating a sample with a saccharide-removing agent, preferably a sample also subjected to deproteinization. The reagent is added in an amount of 0.5 to 10 units, preferably 1 to 5 units of the enzyme per 1 ml of the sample, and is added at 4 to 50 ° C., preferably 25 to
After incubating at 40 ° C. for 0.5 to 3 hours, preferably 0.5 to 1 hour, the amount of hydrogen peroxide produced is then measured with a reagent for detecting hydrogen peroxide, and 1,5-
The amount of AG should be calculated. Next, the present invention will be specifically described with reference to experimental examples and examples. Experimental Example 1 (1,5 using pyranose oxidase
-Calibration curve of AG) A coloring solution prepared by dissolving 1/15 M phosphate buffer (pH 5.6), ABTS and HRP at 4 mM and 12 unit / ml, respectively.
In 5 ml, 0.1 ml of 1,5-AG standard solution, 0.3 ml of 1 / 15M phosphate buffer, 0.1 ml of 5 mg / ml pyranose oxidase (specific activity for glucose 5 unit / mg, manufactured by Takara Shuzo)
Was added and reacted at 37 ° C. for 1 hour. FIG. 1 shows a calibration curve prepared by measuring the absorbance of this reaction solution at 405 nm. Experimental Example 2. (Calibration curve of 1,5-AG using L-sorbose oxidase) Instead of pyranose oxidase of Experimental Example 1, L-sorbose oxidase (specific activity for glucose 4.3 units /
mg) 5mg / ml solution and reacted exactly the same.
A calibration curve for -AG was prepared. The result is shown in FIG. Example (Example of Kit) (1) Preparation of Kit Reagent A: 2.0 ml of a 60% perchloric acid solution was placed in a reagent bottle. Reagent B: PROD 200 mg (5 unit / mg, manufactured by Takara Shuzo), H
RP 0.24mg (100unit / mg, Wako Pure Chemical) and ABTS 220
mg (Boehringer) was dissolved in 100 ml of 0.27 M sodium phosphate buffer (pH = 5.6) so that it was contained in one reagent bottle, and freeze-dried by a conventional method. Pretreatment column: In a reservoir (1.5 ml container, manufactured by Analytical International) equipped with a frit filter, AG50W-X8 (H type) 0.1 ml and A in order from the bottom.
G1-X8 (OH type, both manufactured by Bio-Rad) 0.4m
l was filled. Further, a frit filter was attached from above the filled resin, and fixed so that the resin did not move. Also,
A cap was attached to the outlet, and the inlet was sealed with a seal to prevent the filled resin from drying or absorbing carbon dioxide gas in the air and deteriorating. Standard solution: 0.2 ml of a 1 mg / ml 1,5-AG solution was placed in a reagent bottle and freeze-dried by a conventional method. (2) Procedure 200 μl of a serum sample was placed in an Eppendorf tube (1.5 ml capacity), 15 μl of reagent A was added, and the mixture was stirred. 100 μl of the centrifuged supernatant was removed from the cap and seal, and charged to a pretreatment column set on a plastic tube. After the liquid has completely passed through the resin, 0.5 ml of distilled water
Was added and washed. Further, the mixture was washed twice by 0.5 ml each time, and 1.6 ml of the liquid passed through the pretreatment column was recovered. 100 ml of distilled water was added to the bottle of Reagent B, and the solution was reconstituted. After passing through a chip equipped with a filter to remove suspended matter, 0.5 ml of the solution was added to the above passing solution, followed by stirring and incubation at 37 ° C. for 1 hour. . The absorbance at 405 nm of this reaction solution was measured with a conventional spectrophotometer. Also, 5 ml of distilled water was added to the bottle of the standard solution to prepare a standard solution (40 μg / ml), which was further diluted 2-fold, and a calibration curve was prepared with three points of distilled water. Was determined from the absorbance value of The calibration curve was measured by adding 1.5 ml of distilled water and 0.5 ml of a reconstituted solution of reagent B to 100 μl of the above sample, stirring and reacting in the same manner as for the sample, and the absorbance was measured. The same results as in Reference Example 3 were obtained by the kit method. Reference Example 1. (Measurement of 1,5-AG using pyranose oxidase) Glucose and 1,5-AG were respectively 1 mg / ml and 0.1 mg / ml.
A model test solution containing a concentration of ml was prepared. Glucose was removed by applying the pretreatments shown in 1 to 5 below, and the remaining 1,5-AG was measured by the method shown in Experimental Example 1. The quantification of 1,5-AG was performed by
The standard solution was pretreated in exactly the same manner as the model test solution, and the calibration was performed using a calibration curve created. The results are shown in Table 1. (1) (Method of removing a substrate other than 1,5-AG as a complex with boric acid) 0.02 ml of a 0.8 M aqueous solution of boric acid is added to 0.2 ml of the test solution and stirred. 0.1 ml of the supernatant of the treated solution was passed through a column packed with 0.5 ml of an anion exchange resin AG1-1X8 (manufactured by Bio-Rad) in the form of OH, washed with 1.5 ml of distilled water and passed through the column.
Get 1.5ml. (2) (Method of adsorbing and removing a substrate other than 1,5-AG with a strongly basic anion exchange resin) 0.2 ml of a test solution is subjected to an OH type anion exchange resin AG1-X8.
(Biolat 400 mesh product) After passing through a column filled with 0.5 ml, washed with 1.5 ml of distilled water and passed through 1.5 ml
Get. (3) (Method of decomposing and removing substrates other than 1,5-AG with hydrochloric acid) 0.25 ml of 36% hydrochloric acid was added to 0.2 ml of the test solution, and the solution was sealed and sealed at 110 ° C.
For 1 day. The reaction mixture is evaporated to dryness to remove hydrochloric acid, and redissolved by adding 0.2 ml of distilled water. 0.1 ml of this supernatant was used for the acetic acid type anion exchange resin AG1-X.
After passing through a column packed with 80.4 ml and 0.2 ml of H-type cation exchange resin, the column is washed with 1.5 ml of distilled water to obtain 1.5 ml of a permeate. (4) (Method of reducing substrates other than 1,5-AG with sodium borohydride) To 0.5 ml of the test solution, 0.05 ml of a 40 mg / ml sodium borohydride aqueous solution was added, and the mixture was added at 37 ° C for 30 minutes. react. To decompose the sodium borohydride remaining after the reaction, 0.005 ml of a 60% strength aqueous solution of perchloric acid is added to adjust the pH to 5-6.
0.1 ml of 1.8% barium hydroxide was added to 0.1 ml of the supernatant of the reaction solution.
0.2 ml of hydrate aqueous solution and 0.2 ml of 2% zinc sulfate heptahydrate
, And the mixture is stirred and centrifuged at 3,000 rpm for 10 minutes to obtain a reduction treatment liquid from which precipitates are removed. (5) (Method of oxidizing glucose with glucose oxidase) 0.2 ml of a 100 unit / ml glucose oxidase solution (manufactured by Boehringer) is added to 0.4 ml of a test solution and reacted at 37 ° C for 1 hour. 0.2 ml of a 0.1 mg / ml catalase solution (manufactured by Sigma) is added to the reaction solution, and the mixture is reacted at 37 ° C. for 5 minutes to completely decompose hydrogen peroxide generated by the glucose oxidation reaction. Next, the mixture was heated in boiling tow water for 5 minutes to inactivate and remove glucose oxidase and catalase.
A glucose oxidase-treated supernatant is obtained by centrifugation at 00 rpm for 10 minutes. (6) (Method of phosphorylating glucose with hexokinase) 0.2 ml of an aqueous solution of 1.8% barium hydroxide octahydrate in 0.1 ml of a test solution, and 2% aqueous solution of zinc sulfate / 7 hydrate 0.2m
After adding l and stirring, centrifuge at 3000 rpm for 10 minutes to obtain the protein-free supernatant. To 0.2 ml of this supernatant, add 0.1 M phosphate buffer (pH
7.0) 0.64 ml, 0.2 M magnesium chloride solution 0.1 ml, 0.2
Add 0.05 ml of MATP solution and 0.01 ml of 1400 unit / ml hexokinase solution (manufactured by Boehringer).
A minute reaction is performed to obtain a treatment liquid. Reference Example 2. (Measurement of 1,5-AG using L-sorbose oxidase) A model test solution exactly the same as in Example 1 was subjected to the same pretreatment as in Example 1 to remove glucose, and the remaining 1,5 was removed. -AG was measured by the method of Experimental Example 2. Table 1 shows the measurement results. [Table 1] Reference Example 3 (Measurement of 1,5-AG in Serum Using Pretreatment Column) Ion Exchange Resin AG50W-X8H Form, AG1-X8OH
Form, AG1-X8 boric acid form (both manufactured by Bio-Rad Co., Ltd.) were packed in small columns of 0.3 ml, 0.5 ml and 0.2 ml, respectively, in order from the bottom to form a sample pretreatment column. To 0.2 ml of human serum, 15 μl of a 60% aqueous solution of perchloric acid was added, shaken, centrifuged to remove proteins, and 0.05 ml of the resulting supernatant was passed through the sample pretreatment column described above.
After washing with 3 ml of distilled water, 3 ml of a passing solution was obtained. Next, 3 ml of this passing solution was concentrated to dryness and redissolved in 0.25 ml of distilled water to obtain a treatment solution for the pretreatment column. 1,5-residue remaining in the processing solution
AG was measured by the method shown in Experimental Example 1. That is, quantification was performed using a calibration curve prepared using a 1,5-AG standard substance. FIG. 3 shows the correlation between the serum 1,5-AG values of normal and diabetic patients thus measured and the serum 1,5-AG values when the same sample was measured by the gas chromatography method. As is clear from this figure, the quantitative value by the method of the present invention and the quantitative value by the gas chromatography showed a correlation.
The same results as in this example were obtained by following the procedure of this example except that Boric acidgel was used instead of the AG1-X8 boric acid form. Reference Example 4 (Measurement of 1,5-AG in urine using a pretreatment column) Centrifon-10 ultrafiltration device Centricon-10 (manufactured by Amicon)
A 10 ml human urine sample, which had been deproteinized using the above method, was passed through 1 ml of the ion exchange resin AG50W-8XH form and 1 ml of the AG1-X8 acetic acid form in that order, and washed with 6 ml of distilled water to obtain a desalted urine sample. This is evaporated to dryness and distilled water 0.5m
After redissolving with l, the solution was passed through the pretreatment column described in Example 3 and washed with 3 ml of distilled water to obtain 3.5 ml of a liquid passed through the pretreatment column. Next, this treated solution was concentrated to dryness and redissolved in 0.2 ml of distilled water to obtain a sample for 1,5-AG measurement. 1,5
-AG was measured in Example 1 using 0.1 ml of the above sample.
The method was performed in the manner described above. Also, 1,5 remaining for comparison
-The sample for AG measurement was quantified using a gas chromatograph. Table 2 shows the results. [Table 2] Reference Example 5 (Measurement of 1,5-AG in Serum Using Pretreatment Column Made of Boric Acid-Binding Resin) H-form of ion-exchange resins AG50W-X8, OH-form of AG1-X8, boric-acid form of AG1-X8 (all 400 mesh product, Bio-Rad), 0.3ml and 0.5m respectively
The sample pretreatment column was prepared by sequentially packing each small column of 0.2 ml each in a layered manner from the bottom. After adding 0.2 ml of human serum to 15 μl of a 60% aqueous solution of perchloric acid and shaking, centrifugation is performed to remove proteins. 0.1 ml of the obtained supernatant is passed through the sample pretreatment column and distilled water is added. Wash with 3 ml and pass through 3.
1 ml was obtained. Next, 3.1 ml of this passing solution was concentrated to dryness,
1.0 ml of 1,5-AG detection reagent was added and reacted at 37 ° C. for 1 hour. The absorbance of this reaction solution at 405 nm was measured and quantified by a standard curve prepared by treating a 1,5-AG standard solution in the same manner as in the case of human serum. In addition,
1,5-AG detection reagent is PROD2.5unit / ml, HRP
It is a 1/15 M phosphate buffer (pH 5.6) containing 60 m unit / ml and 1 mM ABTS. A good correlation was observed between the thus-measured serum 1,5-AG values of normal and diabetic patients and the serum 1,5-AG values when the same sample was measured by gas chromatography. Was. Reference Example 6 (Measurement of 1,5-AG in Serum Using Pretreatment Column Made of Strongly Basic Anion Exchange Resin) H Form of Ion Exchange Resin AG50W-X8, OH Form of AG1-X8 (Each is a 400 mesh product, (Manufactured by Bio-Rad Co., Ltd.) were sequentially packed in layers from the bottom into small columns of 0.1 ml and 0.4 ml each to form a sample pretreatment column. 15 ml of an aqueous solution of 0.2 ml to 60% perchloric acid in human serum was added, and the mixture was immediately shaken, followed by centrifugation to remove proteins. 0.1 ml of the obtained supernatant was passed through the column for sample pretreatment described above, and washed with 1.5 ml of distilled water to obtain 1.6 ml of a passing solution. Next, the passed liquid was concentrated to dryness, and quantified in the same manner as in Reference Example 3 below. The same result as in Reference Example 3 was obtained also by this method. Reference Example 7 Experimental example 1 for nitrocellulose membrane (pore size 1 μm) by a conventional method
Immobilized pyranose oxidase. That is, pyranose oxidase (specific activity for glucose 5 unit / m
g, manufactured by Takara Shuzo), 5 mg and 5 mg bovine serum albumin 1/15
Dissolve it in 0.5 ml of M phosphate buffer (pH = 7.2), add 0.1 ml of 1% glutaraldehyde solution, pour this solution on a nitrocellulose membrane (pore size 1 μm, diameter 2 cm, 5 sheets) and air dry overnight. 1 / 15M phosphate buffer (pH = 6.0) 10ml
It was washed with to obtain a nitrocellulose membrane on which pyranose oxidase was immobilized. This pyranose oxidase membrane was attached to the surface of a hydrogen peroxide electrode (manufactured by Ishikawa Seisakusho), and 1,5-AG standard solution (1,5-AG concentration: 1,4,16,3
2 μg / ml) 0.1 ml was passed under the following conditions, the amount of hydrogen peroxide generated was measured as the area of the obtained peak, and a calibration curve was prepared. Mobile phase: 1/15 M phosphate buffer (pH = 5.6) Mobile phase flow rate: 0.5 ml / min Temperature: 25 ± 1 ° C. Next, 3 ml of the liquid passed through the sample pretreatment column obtained in Reference Example 3 A solution obtained by dissolving the concentrated and dried product in 0.25 ml of a 1/15 M phosphate buffer (pH = 5.6) was applied to the electrode provided with the pyranose oxidase membrane in the same manner as in the case of the 1,5-AG standard solution. After passing through, the area of the obtained peak was measured, and 1,5-AG was quantified from the calibration curve. The correlation between the serum 1,5-AG value of the normal human and the diabetic patient thus measured and the serum 1,5-AG value when the same sample was measured by the gas chromatography method was examined. Similarly, both showed a correlation. EFFECTS OF THE INVENTION According to the present invention, there is no need for complicated labeling as is conventionally done by gas chromatography, maintenance and management of high-level analytical equipment are not required, and a large number of cases are required. It became possible to quantify 1,5-AG in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: 1,5-A using pyranose oxidase
Calibration curve of G [Figure 2] 1,5 using L-sorbose oxidase
-Calibration curve of -AG Fig. 3 shows the enzymatic method and the gas chromatography method when the 1,5-AG value in serum was measured.

   ────────────────────────────────────────────────── ─── Continuation of front page                   (56) References JP-A-63-185397 (JP, A)                 BICHIMICA ET BIOP               HYSICA ACTA, 167 (1968)               P. 501-510

Claims (1)

(57) [Claims] An enzyme reagent for quantifying 1,5-anhydroglucitol containing pyranose oxidase or L-sorbose oxidase.