JPH0662846A - Stabilization of enzyme for analytical reagent and enzyme for solution-state analytical reagent - Google Patents

Abstract

PURPOSE:To provide an enzyme for an analytical reagent sufficiently stable in a solution state. CONSTITUTION:An enzyme for an analytical reagent selected from peroxidase (POD), uricase, cholesterol oxidase (COD), ascorbic acid oxidase (AsOD) and lipoprotein lipase (LPL) is chemically bonded to a water-soluble carrier selected from bovine serum albumin, dextran and polyethylene glycol so as to be improved in stability in a solution state. Consequently, the analytical reagent can be provided in a solution state.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technique for stabilizing an enzyme for an analytical reagent. In health care and diagnosis of diseases, various tests including analysis of blood components are carried out. There are many methods using enzymes for the analysis of blood components, but some enzymes are unstable, and stabilization of the enzymes is an important issue in order to improve the reliability of the analysis. Further, stabilization of the enzyme is an unavoidable problem in order to supply the analysis reagent, which is often lyophilized in the form of distribution, in a solution state.

[0002]

Problems of the prior art Freeze-drying is the most common method for storing active proteins such as enzymes. Enzymes for analysis reagents are no exception, enzymes, coenzymes, buffer components,
The components necessary for analysis such as color-developing components are mixed in advance and freeze-dried before being supplied. This lyophilized composition can be made into a reagent solution for analysis by dissolving it in a predetermined amount of water at the time of use. In freeze-drying the enzyme reagent for analysis, a stabilization technique is known in which a high molecular compound such as sucrose, mannitol, galactose, albumin, or a substrate or reaction product of an enzyme is allowed to coexist. By using these substances, stabilization in the freeze-dried state can be expected to some extent.

Recently, however, it has been desired to supply the enzyme reagent in a solution state. The reason for this is that the freeze-dried reagent requires time and effort to dissolve in water, and there is concern about the stability after dissolution. When dissolved in water, the performance of the reagent cannot be ensured unless the solidified components are completely dissolved. On the other hand, if the mixture is vigorously stirred, the enzyme protein may be inactivated by foaming, or the foam on the liquid surface generated by the surfactant may interfere with the quantitative sampling of the reagent. Further, if the stability after dissolution is not guaranteed, the once dissolved reagent must be used up in a short time, which is economically disadvantageous.

Under these circumstances, it is desired to supply an enzyme reagent for analysis in a solution state, but a sufficient stabilization technique has not been established for many enzymes. Among them, peroxidase (hereinafter abbreviated as POD), which constitutes a hydrogen peroxide coloring system adopted as a coloring system in many analytical reagents, uricase necessary for measuring uric acid, and cholesterol oxidase used for measuring cholesterol (hereinafter CO
The amount of ascorbate oxidase (hereinafter abbreviated as AsOD) used for measurement and elimination of ascorbic acid and the lipoprotein lipase (hereinafter abbreviated as LPL) used for measurement of triglyceride are relatively large. Therefore, the effect of stabilization is particularly great in terms of economics, but there is still room for improvement in stability in a solution state.

Regarding the stabilization of the enzyme by binding to a water-soluble carrier, an example in which polyurethane is bound to glucosidase for stabilization (Japanese Patent Publication No. 3-36511) is known, but for other analytical reagents. There are no reports on enzymes. In general, polyethylene glycol (hereinafter PEG
It is known that a water-soluble carrier such as () is used for reducing the antigenicity of the enzyme preparation in vivo and prolonging its half-life. There are no reports of effectiveness.

[0006]

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a technique for imparting sufficient stability in a solution state to five kinds of enzymes which are relatively frequently used among enzymes for analytical reagents. Is. In the present invention, the five types of enzymes to be stabilized include POD, uricase, COD, and A.
sOD and LPL. It is an object of the present invention to provide a stabilization technique that makes it possible to distribute these enzymes for analytical reagents in a solution state.

[0007]

According to the present invention, an enzyme selected from POD, uricase, COD, AsOD and LPL is a water-soluble enzyme selected from bovine serum albumin (hereinafter abbreviated as BSA), dextran and PEG. A method for stabilizing an enzyme for an analytical reagent in a solution state, which is characterized by being chemically bonded to a carrier, and a solution-like enzyme for an analytical reagent stabilized by this method.

PO to be stabilized in the present invention
As D, uricase, COD, AsOD, and LPL, those used for general analytical reagents currently distributed as freeze-dried products can be used as they are. POD is derived from horseradish, uricase is derived from microorganisms such as Candida, COD is derived from microorganisms such as Nocardia, and AsOD is derived from plants such as squash and cucumber. As LPL, those derived from microorganisms such as Chromobacterium are well known. The stabilizing effect of the present invention can be obtained by chemically bonding a water-soluble carrier selected from BSA, dextran and PEG to these enzymes.

At this time, it is necessary to take care so that the activity of the enzyme is not hindered by the binding with the water-soluble carrier. Preferred binding methods include, for example, a method of activating the enzyme with glutaraldehyde and then reacting it with a water-soluble carrier, or a method of activating the water-soluble carrier with periodate or the like to bind the enzyme. By using such a binding method, it is possible to stabilize the enzyme without losing its activity. When glutaraldehyde is likely to inactivate the enzyme, a method of activating the water-soluble carrier with glutaraldehyde may be adopted.

The enzyme for an analytical reagent stabilized by binding to a water-soluble carrier may be dissolved in a suitable buffer together with other enzyme components such as other enzymes, coenzymes, color formers, etc. to prepare an analytical reagent. it can. The following is an application example of the enzyme for a stabilized analytical reagent of the present invention. Stabilized POD: All hydrogen peroxide coloring system Stabilized uricase: Uric acid measurement system Stabilized COD: Cholesterol measurement system Stabilized AsOD: Ascorbic acid measurement and elimination Stabilized LPL: Triglyceride measurement Stabilization analysis of the present invention Other known stabilizers may be added to the reagent enzyme. Surfactants, organic solvents, chelating agents and the like are known as such known stabilizers.

[0011]

The BSA, dextran and PEG used in the present invention are POD, uricase, COD, AsOD and L.
It has the effect of stabilizing these enzymes in solution by binding to PL. Although the principle of stabilization is unknown, it is presumed that the binding to the water-soluble carrier protects the hydrogen and hydrophobic bonds of the enzyme protein to prevent structural changes and enhances the stabilizing action.

[0012] These water-soluble carriers function as stabilizers by the action of protecting the three-dimensional structure of the protein even when they are simply allowed to coexist with the protein. However, the simple coexistence greatly limits the opportunity for the two to effectively approach each other when made into an aqueous solution, and as a result, a sufficient stabilizing action cannot be obtained. On the other hand, in the present invention, by chemically binding the water-soluble carrier to the enzyme, a large stabilizing effect that cannot be realized by simple coexistence is produced. Of course, it cannot be denied that the water-soluble carrier in the present invention will exhibit the same action as when it is simply allowed to coexist. However, the present invention utilizing the new effect of stabilization by chemical bond between the enzyme and the water-soluble carrier is clearly distinguished from the stabilization technique by simply mixing the enzyme and the water-soluble carrier. Next, the present invention will be described in more detail based on examples.

[0013]

【Example】

1-1. Preparation of Stabilized POD Stabilized POD was prepared by coupling with BSA. POD (derived from horseradish, manufactured by Toyobo) 10
0.1 M phosphate buffer (pH 6.8, hereinafter abbreviated as PBS) containing 1.25% glutaraldehyde 0.2 mg
It was dissolved in ml and reacted at room temperature for 18 hours. After the reaction, 0.
Sephadex G-10 equilibrated with 15 M NaCl
It was adsorbed on the No. 0 column and eluted with the same NaCl solution to separate each protein fraction to obtain an activated POD. 1 ml 0.15M
Activated POD in 5 mg BSA dissolved in NaCl
Mix 1 ml, 0.1 ml of 1M carbonate buffer (pH 9.5)
Was added and mixing was continued at 4 ° C. for 24 hours. Further, 0.1 ml of 0.2 M lysine was added and mixed at 4 ° C for 2 hours, and the finally obtained reaction solution was dialyzed against physiological saline at 4 ° C to obtain about 2.5 ml of a stabilized POD solution. It was

1-2. Confirmation of Stabilizing Action The stability of the stabilized POD obtained in 1 and the POD before the treatment were compared. Prepare a reagent for measuring hydrogen peroxide having the following composition,
The sample was stored at 2 to 10 ° C and the change in POD activity with time was observed. The activity is 3.0 ml with 0.05 ml of 12 mM hydrogen peroxide.
It was determined by reacting ml of a reagent for measuring hydrogen peroxide at 37 ° C. for 10 minutes, and measuring the absorbance at 505 nm after the reaction was completed. The results are shown in Fig. 1. It was confirmed that the POD stabilized by the present invention retains sufficient activity for 6 months or more when stored at 2 to 10 ° C. On the other hand, in the POD before treatment, the activity was already less than half of the activity at the time of preparation in the next month, and most of the activity was lost after 3 months. Reagent for measuring hydrogen peroxide: Stabilized POD 1 ml (or POD 4 mg before treatment) 4-aminoantipyrine 10 mg Phenol 40 mg Dissolve with 0.1 M PBS to make the total volume 100 ml.

2-1. Preparation of Stabilized Uricase A uricase stabilized by coupling with dextran was prepared. 50 mg of dextran were reacted with 1.0 ml of 0.06 M NaIO ₄ solution for 30 minutes at room temperature.
Thereafter, 1.0 ml of 0.16 M ethylene glycol was added, the reaction was stopped by standing at room temperature for 1 hour, and the mixture was dialyzed against 0.01 M sodium carbonate buffer (pH 9.5) at 4 ° C. overnight. To the reaction solution after dialysis, 5 mg of uricase (derived from Candida, manufactured by Toyobo Co., Ltd.) was added and reacted at room temperature for 2 hours,
Add 5 mg of sodium borohydride and add 12 at 4 ° C.
After reacting for a period of time, it was dialyzed against physiological saline at 4 ° C. for 14 hours to obtain about 2.4 ml of stabilized uricase.

2-2. Confirmation of stabilizing effect The stability of the stabilized uricase obtained in 2-1 was compared with that of the uricase before treatment. A reagent for measuring uric acid having the following composition was prepared and stored at 2 to 10 ° C, and changes in uricase activity with time were observed. The activities were 2.mM uric acid 0.05 ml and 3.
React with 0 ml of reagent for measuring uric acid at 37 ℃ for 10 minutes, and
It was determined by measuring the absorbance at 55 nm. It was confirmed that the uricase stabilized by the present invention maintains a sufficient activity for 6 months or more when stored at 2 to 10 ° C. Uric acid measurement reagent: Stabilized uricase 1 ml (or uricase 2 mg before treatment) Stabilized POD (Example 1) 1 ml 4-aminoantipyrine 10 mg EMST 140 mg Dissolved in 0.1 M PBS (pH 6.5) to a total volume of 100 m
EMST is an abbreviation for N-ethyl-N- (β-methylsulfonamidoethyl) m-toluidine sodium

3-1. Preparation of Stabilized COD A COD stabilized by conjugation with PEG was prepared. PEG6000 (Wako Pure Chemical Industries, Ltd.) 2.0g
Was dissolved in 10 ml of dioxane, 1 g of 1,1′-carbonyldiimidazole was further added and mixed, and the mixture was reacted at 37 ° C. for 90 minutes. After the reaction, the mixture was dialyzed against 0.15 M NaCl overnight and freeze-dried to obtain about 2 g of activated PEG.
150 mg of the obtained activated PEG and 20 mg of COD (derived from Nocardia, manufactured by Boehringer Mannheim) were added to 0.1
It was dissolved in 2 ml of M PBS (pH 7.0) and reacted at 4 ° C. for 20 hours. After the reaction, it was adsorbed on a Sephadex G-200 column equilibrated with 0.15 M NaCl, and eluted with the same NaCl solution to separate each protein fraction to stabilize COD.
About 2.4 ml was obtained.

3-2. Confirmation of Stabilizing Action The stability of the stabilized COD obtained in 3-1 and the stability when COD was simply allowed to coexist with PEG were compared. Prepare a cholesterol measuring reagent with the following composition and store it at 37 ° C for COD.
The change in activity over time was observed. For the activity, 3.0 ml of a reagent for measuring cholesterol was added to 0.05 ml of cholesterol 300 mg / dl (isopropanol solution), and the mixture was reacted at 37 ° C. to take a time course (555 nm). The result is shown in Figure 2.
As shown in. When coexisting with dextran, an extreme decrease in activity is observed 2 weeks after preparation, but COD stabilized by the present invention shows almost no change in activity in the same period. Cholesterol measurement reagent: Stabilized COD 1 ml (or untreated COD 8 mg + PEG 60 mg) Stabilized POD (Example 1) 1 ml 4-aminoantipyrine 10 mg TOOS 40 mg Dissolve with 0.1 M PBS (pH 6.0) and total 100 m
TOOS is an abbreviation for N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium

4-1. Preparation of Stabilized AsOD Stabilized AsOD was prepared by binding with BSA. 20 mg of AsOD (derived from pumpkin, manufactured by Boehringer Mannheim) was dissolved in 0.5 ml of 0.1 M PBS (pH 7.0) containing 1.0% glutaraldehyde and left at room temperature for 18 hours. After the reaction, 0.15M N
It was adsorbed on a Sephadex G-200 column equilibrated with aCl, and eluted with the same NaCl solution to separate each protein fraction to obtain activated AsOD. 1 ml of 0.15M NaCl
Activated AsOD 1 ml in which 20 mg of BSA was dissolved in
Were mixed, 0.1 ml of 1M carbonate buffer (pH 9.5) was added, and the mixture was reacted at 4 ° C. for 24 hours. Further, 0.1 ml of 0.2 M lysine was added and mixed at 4 ° C. for 2 hours, and the finally obtained reaction solution was dialyzed against PBS (pH 6.5) at 4 ° C. to stabilize AsOD of about 2. 5 ml was obtained.

4-2. Confirmation of Stabilizing Action The stability of the stabilized AsOD prepared in 4-1 was measured by simply using BS.
It was compared with the case of coexisting with A. AsO with the following composition
A D-containing solution was prepared and stored at 37 ° C. together with the stabilized AsOD obtained in 4-1 to compare its stability. Activity is
0.1 ml of 100 mg / dl ascorbic acid solution in 0.1 ml of the stabilized AsOD or AsOD-containing solution obtained in 4-1.
Was added, the mixture was reacted at 37 ° C. for 5 minutes, and determined from the change in absorption of ascorbic acid at 240 nm. The results are shown in FIG. Stabilized AsOD retained its ability to eliminate ascorbic acid for over a week, whereas when simply mixed with BSA, most of the activity was lost the day after dissolution. AsOD-containing solution: AsOD 8 mg BSA 8 mg 0.1 M PBS (pH 6.5) to make the total volume 1.0 ml.

5-1. Production of stabilized LPL LP stabilized by binding with dextran
L was produced. LPL (Ch
45 mg of romobacterium-derived Asahi Kasei) was used to obtain about 2.6 ml of stabilized LPL. Stabilized L thus obtained
PL has excellent stability in a solution state as compared with untreated PL or LPL simply coexisting with dextran.

[0022]

According to the present invention, POD, uricase,
COD, AsOD, LPL can be highly stabilized in solution. The enzyme for an analytical reagent stabilized in the present invention maintains a practical activity for 6 months or more even at a temperature of 2 to 10 ° C. which is convenient for transportation. As described above, the present invention greatly contributes to the supply of the analytical reagent in a solution state. The enzyme targeted for stabilization in the present invention consumes a relatively large amount among many enzyme reagents. Therefore, the effect of the present invention is great from an economical point of view.

[Brief description of drawings]

FIG. 1 is a POD stabilized by the present invention.
2 is a graph showing the result of comparing the stability of POD before treatment. In the figure, the vertical axis represents POD activity immediately after preparation of the reagent, which is 1
The value of 00 is set, and the horizontal axis indicates time (month).

FIG. 2 is a COD stabilized by the present invention.
2 is a graph showing the results of comparing the stability when untreated COD is simply allowed to coexist with PEG. In the figure, the vertical axis represents the amount of change in absorbance at 555 nm, and the horizontal axis represents the reaction time (minutes).

FIG. 3 is AsOD stabilized by the present invention.
2 is a graph showing the results of comparing the stability when untreated AsOD was simply allowed to coexist with BSA. In the figure, the vertical axis shows the value with the AsOD activity (ascorbic acid scavenging ability) immediately after reagent preparation as 100, and the horizontal axis shows the storage period (days).

Claims (2)

[Reference Number] P-000260 [Claims] 1. An enzyme selected from peroxidase, uricase, cholesterol oxidase, ascorbate oxidase and lipoprotein lipase is chemically bonded to a water-soluble carrier selected from bovine serum albumin, dextran and polyethylene glycol. Method for stabilizing enzyme for analytical reagents in solution 2. An enzyme selected from peroxidase, uricase, cholesterol oxidase, ascorbate oxidase and lipoprotein lipase is chemically bonded to a water-soluble carrier selected from bovine serum albumin, dextran and polyethylene glycol. Stabilized solution enzyme for analytical reagents