JPS61234783A - Improved method of determining liquid component - Google Patents

Abstract

PURPOSE:To make response rate of enzymatic reaction high and to stabilize enzymatic activity even by continuous use of many times, by using a specific polymer as a carrier for an immobilized enzyme to be used for quantitative analysis of urea nitrogen in liquid, uric acid, etc. CONSTITUTION:In an immobilized enzyme to be used for quantitative analysis of urea nitrogen, uric acid or creatinine contained in liquid, urease, uricase or creatinine deaminase is used as an enzyme and a water-insoluble amino group-containing polyacrylonitrile polymer or its derivative is used as a carrier for immobilizing it. It is preferably in a fibrous state.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved method for the determination of liquid components containing urea nitrogen, uric acid, and creatinine.

In recent years, enzyme analysis methods that use enzymes to quantify and analyze components contained in liquids have been developed and have come to be widely used. However, this enzyme analysis method is uneconomical since it consumes a large amount of expensive enzymes, and has disadvantages such as the operation is still cumbersome, the measurement time is long, and the enzyme is unstable. Ta. Therefore, in order to improve these shortcomings, immobilized enzymes are attracting attention, and in particular, so-called enzyme electrodes that are combined with this Lft electrochemical measurement method are attracting attention.Next, measurement methods using this enzyme electrode. The method is simple to operate, requires fewer reagents, shortens the primary side fixation time, and significantly reduces the amount of enzyme consumed.It has many advantages, and many enzyme electrodes have been reported. 0 In addition, these enzyme electrodes are attracting a lot of attention, especially in the field of clinical chemistry, and are also attracting attention in the fermentation, food fields, etc., for analysis of raw materials, product substances, and products.0 Furthermore, ultrafiltration membranes and So-called liquid component monitoring systems that continuously measure the components of biological fluids such as dialysate and fermentation fluid using hollow fibers, such as the artificial pancreas monitoring system for blood sugar measurement (product name: BIO)
8TATOR) and artificial kidney monitoring systems (Fermentation and Industry, Vol. 35, No. 11, pp. 42-47; Journal of the Japan Clinical Laboratory Automation Research Society, Vol. 2, No. 1, pp. 24-27) have attracted attention. There is.

This artificial kidney monitoring system is for measuring urea nitrogen, uric acid, and creatinine in patients with kidney disease.In other words, patients with uremia or nephritis have high levels of urea nitrogen, uric acid, and creatinine in their blood. 1 to 2 times a week because it becomes high and can lead to death if not removed promptly.
Artificial dialysis is performed for about 6 hours each time, and the components to be removed are measured by the monitoring system during this dialysis. If the quantitative amount of the component could be measured moment by moment, it would be possible to shorten the dialysis time and eliminate the risk of other harms caused by dialysis.Currently, the only monitoring system on the market is a measuring device that only monitors urea nitrogen. do not have. That is, for this monitor, an immobilized enzyme is used, which is a support such as porous glass beads coated with polymethyl glutamic acid, and urease is immobilized thereon by azidation. ,
Alternatively, a nylon tube is treated under specific conditions to cleave the amide bond, and an immobilized enzyme is used to immobilize the enzyme with glutaraldehyde. For monitoring systems that require continuous use many times and function well over a long period of time, enzymes must be immobilized using a carrier obtained through processing under special conditions. There has been a desire for a method that can satisfactorily measure the liquid components of liquids, and that can also be effectively used for monitoring.

As a result of various studies on enzyme analysis methods that can satisfactorily measure the components of urea nitrogen, uric acid, or creatinine as described above, the present inventors have found that an enzyme that acts as a substrate for urease, uricase, or creatinine deaminase has been immobilized. When the carrier for the immobilized enzyme is a water-insoluble amino group-containing polyacrylonitrile polymer or a derivative thereof that can be easily produced by reducing a polyacrylonitrile polymer, the immobilized enzyme obtained is We discovered that it is stable during the summer, is stable even after repeated use, and has a fast response to enzymatic reactions. We have found that the method can be used in a wide variety of ways, such as in electrochemical measurement units, and have found that urea nitrogen and uric acid creatinine can be measured extremely well.

The present invention has been completed based on the above findings, and is a method for quantifying a liquid component containing urea nitrogen, uric acid, or creatinine using an immobilized enzyme that acts on the component as a substrate. This is an improved method for quantifying liquid components in which the carrier of the immobilized enzyme is a derivative of a water-insoluble amine group-containing polyacrylonitrile polymer.

First, the liquid used in the present invention may be any liquid containing at least one of urea nitrogen, uric acid, and creatinine, such as biological fluids such as blood, plasma, serum, and urine, fermentation liquids, and other liquids containing these. Examples include solutions of reagents containing reagents, preferably liquid samples such as biological fluids in the field of clinical chemistry, and these liquids can be filtered using ultrafiltration membranes (e.g. Diaflow Membrane Plan XM-50) or hollow fibers. It may be a dialysate or filtrate obtained using a filtration device, and in particular, a blood dialysate or filtrate is used as the liquid sample in the above-mentioned monitoring.

The enzymes used to measure these components include, for example, urease to measure urea nitrogen, uricase to measure uric acid, and creatinine deaminase to measure creatinine. They may be selected and combined as appropriate based on the substrate specificity and reaction of the enzyme.

Furthermore, the water-insoluble amine group-containing polyacrylonitrile polymer used in the present invention includes, for example, lOO~
It may be a polyacrylonitrile-based polymer having a porosity of about 10,000 A and containing a water-insoluble amine group and containing at least acrylonitrile as a main component, preferably a polyacrylonitrile system having a porosity of about 100 to 10,000 A. 2) A polymer obtained by reducing a polymer and converting a part of the IJ group into an amino group. In particular, a fibrous form of the enzyme is preferably used as a carrier, since it can immobilize the enzyme at a high titer and has a fast response when the immobilized enzyme is used. In addition, this water-insoluble amino group-containing polyacrylonitrile polymer or fc can be obtained by the method described in Japanese Patent Application No. 53-18001. Polymers, porous fibrous materials, granular materials, or film-like materials of polyacrylonitrile-based polymers containing acrylonitrile as one or the main component are added to and suspended in an insoluble medium such as diethyl ether or dioxane. Add a reducing agent such as 5-tium aluminum hydride and reduce the mixture, usually for 1 to 10 hours, and 2) convert some of the IJ groups to amino groups to obtain a water-insoluble amine group-containing polyacrylonitrile polymer. Goodbye, my friend, the water-insoluble amine group-containing polyacrylonitrile polymer (
The physicochemical properties of the carrier of the present invention (as a porous fibrous material) are as follows.Color properties: It is slightly more yellowish than the porous polyacrylonitrile that is the raw material. The longer the reduction time, the stronger the yellow color becomes.

Viscosity: Unknown (cannot be measured because it does not dissolve in the following solvents) 0 Solubility in solvents: (Compare and list the carrier of the present invention and its raw materials) Carrier Raw material jar DMSO partially dissolved Dissolved DMF "" Concentrated Partially dissolved nitric acid Dissolved 65% KSCN solution I water
Insoluble Insoluble Chloroform Acetonitrile Pyridine to Tromethane cyclohexane diethyl ether dioxane Tetrahydrofuran 30% hydroxide) IJium aqueous solution I It does not completely dissolve. When water is added to this solvent solution, it becomes cloudy, and the following experiment shows that the weight has decreased, indicating that it is partially dissolved.
'C) Support 30 of the present invention obtained at various reduction times
Q, DMF57! After heat treatment at 60°C for 3 minutes, the fibers were separated, washed with water, and their dry weight was measured.The results were as follows.

Reduction time Weight loss rate of carrier: 5 minutes
31.3ch 1 hour 29.0% 5 hours 27.0ch Colorimetry with TNBS (J, Biochem, 81
, 1715 t (1977)]: Although the porous polyacrylonitrile fiber used as a raw material does not exhibit color, the carrier of the present invention exhibits an orange to yellow color, and it was confirmed that it has an amino group.

Amino group content: The amino groups contained in the carrier were confirmed by immobilizing proteins and amino acids on the obtained carrier.

(1) Protein immobilization Support (fibrous material) 181 of the present invention with various reduction times
19 was added to 0.1M borate buffer (p)I8.5) containing 12.5% glutaraldehyde and heated at 0°C.
, the reaction was stirred for 20 minutes, and after the reaction, the temperature was 0.1M.
Wash thoroughly with borate buffer (pH 8.5), then 0.1M phosphate buffer (pH 7.5), and then immediately with 0.1M phosphate buffer (pH 7.5).
Add to 3% bovine serum albumin solution 27 and incubate at 30°C for 1 hour.
Shake the rice for an hour, filter it, collect the liquid, and
The content of bovine serum albumin remaining in the bath was determined by the Lowry method (
J, Biol, chem, , 193, 26
5 (1951)), the amount of bovine serum albumin bound to the carrier was determined by m+.

Further, the carrier was directly added to a bovine serum albumin solution without glutaraldehyde treatment, and after shaking at 30°C for 1 hour, the amount of bovine serum albumin adsorbed onto the carrier was determined in the same manner as above.

The results are shown in Table 1.

(2) Amino acid immobilization The carrier (fibrous material) of the present invention with various reduction times 18
After treating q with a 12.5% glutaraldehyde solution in the same manner as in (1) above, this was treated with o, 3%
Glutamic acid (Gl'u), Threonine (Thr
) containing each amino acid in 0.1 M IJ acid buffer (pH
7,5) It was added to 2 ml and shaken at 30°C for 1 hour. After the reaction, the amino acid content in the solution was quantified using an automatic amino acid analyzer to determine the amount of the amino acid bound to the carrier (in addition, in this test, the amino acid used was The absence of adsorption properties was confirmed by a test using amino acid hydrazide in place of the amino acid).

The results are shown in Table 2.

(3) Amine group content determined by titration of the carrier of the present invention (fibrous material 8079) determined by various reduction times.
i, 2mM hydrochloric acid 1std, and distilled water 3o- for 1 hour, then excess hydrochloric acid was titrated with 2mM sodium hydroxide aqueous solution, and the amino group-containing i- in the carrier was determined as the amount of hydrochloric acid consumed as the hydrochloride. 0 The results are shown in Table 3.

Furthermore, the carrier, which is a water-insoluble polyacrylonitrile polymer containing 7 amino groups, obtained in this way can be used for polyfunctional reagents, such as dialdehydes such as malonaldehyde, succinaldehyde, glutaraldehyde, and adipaldehyde, malones, etc. succinic acid, glutaric acid, adipic acid,
Dicarboxylic acids such as pimelic acid, maleic acid, and phthalic acid; a) Active esters of carboxylic acids such as spalagic acid, glutamic acid, and other amino acids such as ω-amino acids; reactive derivatives such as halides and anhydrides; and their water solubility. Using an appropriate combination of carbodiimide, diamines such as hexamethylene diamine, dodecamethylene diamine, and lysine, a spacer is introduced into the amino group, and the amino group is converted into the terminal active group of the polyfunctional reagent. It may also be used as a derivative of the carrier of the present invention. Further examples of this derivative include α) reacting the carrier of the present invention with glutaraldehyde to introduce it into its amino group; A carrier in which a spacer having a terminal aldehyde group consisting of glutaraldehyde-hexamethylenediamine-glutaraldehyde is introduced into the amino group, (2) a carrier obtained by using lysine in place 9 of the above hexamethylenediamine, (3) ) A carrier having a reactive derivative of a terminal carboxylic acid obtained by reacting a carrier with a reactive derivative of a dicarboxylic acid such as dimethylmalonimidate, dimethylmalonimidate, or dihydrochloride of dimethylmalonimidate, (4) A terminal carboxylic acid is obtained by reacting a dicarboxylic acid anhydride such as succinic anhydride or glutaric anhydride with a carrier, and then reacting it in the presence of N-hydroxysuccinimide, p-nitrophenol fN, or N-dicyclohexylcarbodiimide. /carriers with reactive derivatives of acids, etc., activated with their amine groups in various ways, and derivatives thereof are mentioned, and derivatives of such various carriers are used as advantageous derivatives thereof. The derivatives of carriers may be, for example, fibrous materials with a diameter of 0.1 m or less and lengths of 2 or less, granular materials with a diameter of 0.2 m or less, membrane-like materials with a diameter of 5 to 10 pieces, The immobilization of the present invention using such a carrier or a derivative thereof may be processed into a suitable size, and the shape and size may be adjusted depending on the electrochemical measuring device to be used, which will be described later. To obtain the enzyme, the carrier or derivative thereof and the enzyme to be used are added to an aqueous medium, preferably a buffer solution having a pH that indicates the pH stability of the enzyme to be used, and the mixture is usually incubated at room temperature for 10 minutes or more. By reacting for about 12 hours, the enzyme is immobilized by a covalent bond based on, for example, the amine group in the enzyme molecule and the aldehyde group or carboxyl group introduced into the carrier. Furthermore, as described above, the carrier and the enzyme are reacted in the presence of a polyfunctional reagent such as glutaraldehyde or dimethylazifoimidate without introducing a reactive group such as a terminal reactive derivative into the carrier in advance. Both amine groups are cross-linked, and the carrier and enzyme are further reacted in the presence of a carbodiimide reagent such as 1-ethyl-3-(3-dimethylaminoglopyl)-carbodiimide or other Woodward reagent. An immobilized enzyme may be obtained by bonding the amine group of the carrier and the carboxyl group of the enzyme.

In this way, an immobilized enzyme necessary for measuring urease, uriase, and creatinine deaminase is obtained, and then this immobilized enzyme is used to measure urea nitrogen, uric acid, and other enzymes contained in the liquid. Creatinine is measured and quantified, and various systems are used such as reactor systems using columns of immobilized enzymes and various systems using immobilized enzyme membranes. The electrochemical measuring device used as the next reactor system for measuring the components is as shown in FIG.

That is, in the figure, l indicates a column filled with immobilized enzymes, for example, urease immobilized enzyme for urea nitrogen measurement, uricase immobilized enzyme for uric acid, and creatiny/deaminase immobilized enzyme for creatinine. The amount of these immobilized enzymes used is
Generally 200 U/? for urease? (wet weight 9
That's it, Kuricase 5V? It is sufficient to use about 1 c20 to 200 q (wet weight) of an immobilized enzyme having an enzyme titer of 20 to 200 q (wet weight), and 2 indicates an electrode as an electrochemical measuring section, and a suitable VC is for measuring urea nitrogen. In the case of , an ammonia electrode is used, and in the case of uric acid and creatinine, an oxygen electrode is mentioned, 3 indicates a flow cell, the end of the electrode is connected to this 70-cell, and 4 indicates an inlet for the sample liquid, usually Approximately 5 to 50 μm of the sample liquid may be injected using a microsyringe, and in the case of continuous injection, the sample liquid may be injected continuously. Furthermore, number 5 indicates a buffer tank used in the measurement, and the buffer solution, for example, in the case of measuring urea nitrogen, is a tank containing 2mM EDTA.
025 M phosphate buffer (pH 7.0), 0.05 M borate buffer (pH 8.5) for uric acid, 0.1 M dimethylglutarate buffer (pH 8.0) for creatinine.
,0) or 0.1M dimethylglutarate buffer (pH 8,0) containing 2mM BDTA may be used, and 6 indicates a pump for delivering the buffer, and 7 indicates a constant temperature of the above-mentioned device part. Indicates a constant temperature bath for
The temperature is maintained at 25-40°C, preferably about 37°C. Part 8 is a converter for converting electrochemical changes from the electrode for the recorder shown in part 9. Normally, when the electrode is an oxygen electrode, it is a dissolved oxygen meter, and when the electrode is an ammonia electrode, it is a converter for converting the electrochemical change from the electrode. Amplifiers such as ammonia meters are used. Further, if necessary, data processing devices such as a digital meter (10) and a digital printer (11) may be attached in the figure. Furthermore, when measuring the generated ammonia, such as in the case of urea nitrogen, a tank of alkaline aqueous solution is connected between the immobilized enzyme column and the path of the flow cell, in which the electrodes are all connected, via a valve 12 such as a three-way cock. 13, e.g. 0.2N hydroxide)
It is sufficient to connect a tank containing an IJum aqueous solution. In order to systematically measure multiple components at the same time, for example, when measuring uric acid and urea nitrogen at the same time, first use a 0.05M IJ acid buffer containing 2mM EDTA (pH 7) as the buffer solution. , 5), 1 to 6 in the above figure 1.
In place of 13 and 13, an apparatus as shown in FIG. 2 may be used. Enzyme column, 2bld ammonia electrode,
Reference numeral 13b indicates an alkaline tank for a 0.2 N aqueous sodium hydroxide solution which is connected through a valve 12, and further has a valve 14. If necessary, a three-way cock serving as a waste liquid port may be attached, and 3 is a 70-cell , 4 is the injection port for the sample liquid, 5 is the buffer solution, and 6 is the pump. , all you need to do is attach the necessary converter and data processing device.The electrodes mentioned above are used to measure the components consumed or produced based on the reaction of the enzyme used. For example, when the component to be measured is hydrogen peroxide or carbon dioxide, a hydrogen peroxide electrode or a carbon dioxide electrode may be used as an electrode to measure oxygen, which is a consumed component. For this purpose, a potentiometry type or an ampere type may be used as appropriate. However, as a measuring device using a membrane system, instead of the column of immobilized enzyme in the pre-actor system, an immobilized enzyme membrane is placed between the electrode and the flow cell, preferably in the form of a membrane at the end of the electrode. All you have to do is join them. Furthermore, in these devices, for example, an autosungler, each valve, and a solenoid valve that can electrically control the pump are used to inject the sample liquid,
In addition, a microcomputer is equipped with a channel device for input, etc., for automatic input and control of other attached parts, and a display device that can display elapsed time, device conditions, etc., and an operation panel for input control, etc. The input key panel for the computer may be connected and the computer automation may be performed through an interface.

For subsequent measurements, the pump first transports a certain amount of the necessary buffer solution at a flow rate of, for example, 0.1 to 2-7 minutes to stabilize the readings of the device, and then the injection For example, 5 to 50 μt of sample liquid is injected into the inlet, and this is reacted in a column packed with immobilized enzyme, and then introduced into a flow cell with an internal volume of about 0.1 to 0.5 μm, where the sample liquid is consumed by the reaction. The component fc is used to sense the generated component with its electrode, and can usually be detected 10 to 120 seconds after the liquid is injected. This sensed value may then be converted by a converter, and this value may be recorded or processed as digital data.

As described above, the components of a liquid containing at least one of urea nitrogen, uric acid, and creatinine have been successfully measured and quantified. Although specifically described, the present invention is not limited thereto in any way.

Example 1 In addition to lithium aluminum hydride 3.9e dry diethyl ether 300-, a fibrous material of polyacrylonitrile polymer having a porous structure (manufactured by Asahi Kasei Corporation) 2
．． 52' and then reduced with stirring at 45° C. for 10 hours. Excess IN hydrochloric acid, water, IN hydroxylation after reduction) IJ
A water-insoluble polyacrylonitrile polymer fibrous material (porous) containing 7 amino groups was obtained by sequentially washing with an aqueous solution of aluminum and reducing a portion of the nitrile groups.

The obtained water-insoluble amine group-containing polyacryloni) I
12. Julpolymer fibrous material 300 cm (dry weight).
0.05 M borate buffer containing 5% glutaraldehyde (pH 8.5) 307! and stirred at 0℃ for 20 minutes.
After 0 minutes of reaction, 500 cm of fibrous material (wet m
Add 15 parts of MEDTA (containing 1 mM mercagtoethanol) and stir the reaction at 0°C for 3 hours.
7,5), washed with 0.025 M phosphate buffer containing 2-crystallized DTA to obtain urease-immobilized enzyme (carrier;
Water-insoluble amine group-containing polyacrylonitrile polymer, urease activity 267 U/# (wet weight)].

Next, using this urease-immobilized enzyme, an electrochemical measuring device was prepared as shown in Fig. 1, and fco, that is, 100 my (wet weight) of this urease-immobilized enzyme was placed in a column of the immobilized enzyme (diameter 2.8 30■), and connect the two products (EDTA-containing 0.025 M 1.1 phosphate buffer (pH 7.0) buffer solution tank to the front part of this;
．． An alkaline bath containing 2N sodium hydroxide aqueous solution was connected via a valve to a flow cell (inner volume 0.2 mJ), an ammonia electrode was connected to this flow cell, and these were placed in a constant temperature bath and kept at 37°C. Furthermore, an apparatus was constructed in which a recorder consisting of an ammonia meter (manufactured by Ishikawa Seisakusho) was attached to this ammonia electrode.

The buffer was then applied at a flow rate of 1. At a steady state (after about 8 minutes), urea nitrogen standard solutions of various concentrations, which had been dissolved in the buffer solution and preheated, were added through the injection port.
The immobilized enzyme is injected into the column and reacted in the column to generate ammonium ions, and then a 0.2N aqueous sodium hydroxide solution is added from an alkaline bath to adjust the pH to approximately 12, thereby reducing the ammonia generated. was recorded using an ammonia electrode and an ammonia meter. As a result, in the line 9 shown in Figure 3, the horizontal axis shows the urea nitrogen concentration of the urea nitrogen standard solution,
The vertical axis shows the voltage value obtained by measurement, and a calibration curve showing an extremely good straight line was obtained.

Furthermore, using the above device, under the same conditions, 20 μt of human serum (3 men) was injected through the injection port to measure urea nitrogen in the serum. : Compare it with the value measured using (i-yatron-sha-ri).The results are as shown in the following 7t.

Serum Shop Invention Commercial Product 1 10.2■/dt Lo, 8'l! /dt
2 8.8■/dt 9.2■/dt3
13.8Wi/dt14.3■/dtExample 2■In place of the urease and its solvent in Example 1,
Using uricase (3,2TJA, manufactured by Toyobo Co., Ltd.) 50 ~ and 0.1 M IJ acid buffer (pH 7,5)',
In the same manner, a uricase-immobilized enzyme [uricase activity 8.8 U/p (wet weight)] was obtained.The apparatus for this purpose was as shown in Figure 1. Diameter 2,8 x 30WII+
A column of 1 was filled to form an immobilized enzyme column, and a 0.05 M borate buffer (pH 8,5) tank was connected to the front of this column through a pump, which was then passed through a 70-cell. The flow cell was connected to an oxygen electrode, which was further connected to a recorder via a dissolved oxygen meter.

Furthermore, for the measurement, first, the temperature was kept at 37°C in a constant temperature bath, and the buffer solution was flowed at a flow rate of 1.0 d/min to reach a steady state [8 minute diameter]. The test was carried out by injecting 10 ILL of uric acid standard solution (0.04 (i Li2Co, dissolved in buffer containing).
As shown in the figure, a good calibration curve was obtained.

■ In the same manner as above, after achieving steady state with buffer solution,
Inject a dissolved and preheated uric acid standard solution through the injection port, continue until the measured value on the recorder becomes constant, and then inject another concentration of uric acid standard solution and perform continuous injection measurements. Ta. As a result, a measurement chart as shown in FIG. 5 was obtained (chart speed 1 cm/2 minutes), and based on it, the calibration curve was as shown in FIG.

In addition, it is clear from this measurement chart that the reaction occurs within 60 seconds after injection of the uric acid standard solution, and a constant value is shown in about 100 seconds, making it possible to perform one measurement in an extremely short time. Met.

■ Using the same device as above, instead of the uric acid standard solution, standard serum normal Hyland (5.2 to 5.4 m'i
/dt) and abnormal Hyland (8,7~9.0
'97dl) t 10 μL was injected and measured, and the uric acid content was determined from the above calibration curve. In addition, measurements were made using a commercially available product (Uric Acid/Uricase Color Test: Boehringer), and the results were in good agreement.

Moreover, the measured values are as follows.

This invention Commercially available product Normal Hyland 5.5 my/dt
5.8 mf/dt abnormal Hy land 9
．． 2 W/ dt9.5 */ dl■ Mayu A total of 90 human serum samples were continuously injected (90
As a result, the results shown in Fig. 7 were obtained, which were stable and could be measured continuously.

[Brief explanation of drawings]

FIG. 1 shows the measuring device used in the present invention, in which 1 is an immobilized enzyme column, 2 is an electrode, 3 is a flow cell, 4 is an injection port, 5 is a buffer tank, 6 is a pump, and 7 is a flow cell. Constant temperature bath, 8
1 is a converter, 9 is a recorder, 10 is a digital meter, 11 is a digital printer, 12 is a box, 13 is an alkali bath, and FIG. 2 shows the simultaneous measurement device used in the present invention, and its la and lb are Immobilized enzyme column, 2a and 2b are electrodes, 3 is a flow cell, 4 is an injection port,
5 is a buffer tank, 6 is a pump, 12 is a cock, 13b is an alkali tank, and 14 fl valves; FIG. 3 shows a calibration curve for urea nitrogen measurement; FIG. 4 shows a calibration curve for uric acid measurement; Figure 5 shows a measurement chart 1 for continuous injection in uric acid measurement, and Figure 6 shows a calibration curve based on Figure 5.
Figure 7 shows the stability in uric acid measurement.

Claims (3)

[Claims] (1) In a method for quantifying a liquid component containing urea nitrogen, uric acid, or creatinine using an immobilized enzyme that acts with the component as a substrate, urease, uricase,
An improved method for quantifying liquid components, characterized in that the immobilized enzyme carrier of creatinine deaminase is a water-insoluble amino group-containing polyacrylonitrile polymer or a derivative thereof. (2) The improved method for quantifying a liquid component according to claim 1, wherein the water-insoluble amino group-containing polyacrylonitrile polymer or its derivative is a fibrous material. (3) The improved method for quantifying a liquid component according to claim 1 or 2, wherein the liquid component is a biological fluid.