JP2000083661A - Stabilization of bilirubin oxidase and reagent composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently stabilize bilirubin oxidase used as a regent for clinical examinations, etc., by adding a pentacyano iron complex, etc., and a tertiary amine to a solution containing the bilirubin oxidase. SOLUTION: This method for stabilizing bilirubin oxidase comprises adding a pentacyano iron complex and/or a hexacyano iron complex and a tertiary amine to a solution containing the bilirubin oxidase. The pentacyano iron complex includes a pentacyano aquo iron (II) acid salt of formula I [M is an alkali(ne earth) metal; (n) is 2-5; X is H2O, a halogen, SO4, CO, NH3, etc.], and is preferably contained in an amount of 2-1,000 μM. The hexacyano iron complex includes hexacyano iron (II) acid sodium salt of formula II [M is an alkali(ne earth) metal; (n) is 2-5] and is preferably contained in an amount of 2-1,000 μM. The tertiary amine includes N,N-bis(2-hydroxyethyl)glycine and is preferably contained in an amount of 0.01-1,000 μM. A carboxyl group-containing saccharide (for example, glucuronic acid) is further added in an amount of 25-1,000 μM.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stabilization method capable of stably maintaining the enzyme activity of bilirubin oxidase (hereinafter abbreviated as BOD) for a long period of time, and a reagent to which this stabilization technique is applied. It relates to a composition.

[0002]

2. Description of the Related Art BOD is an enzyme that catalyzes the reaction of oxidizing bilirubin to biliverdin.
It is used for optical measurement of bilirubin contained in biological samples such as serum. Measurement of bilirubin in serum samples
It has a very important clinical significance as an index for diagnosing liver function and hematopoietic function diseases, and is one of the most universal and indispensable measurement items of clinical tests.

[0003] Bilirubin present in serum includes conjugated bilirubin bound to glucuronic acid (direct bilirubin).
And unconjugated bilirubin bound to albumin (indirect bilirubin). In the field of clinical examination, it is known that direct bilirubin in blood is useful as a diagnostic index for obstructive jaundice, and indirect bilirubin is useful as a diagnostic index for hemolytic jaundice and hemolytic anemia. Furthermore, for the diagnosis of these diseases, total bilirubin, which is the total amount of direct bilirubin and indirect bilirubin, has been separately measured.

In general, BOD contained in a reagent is relatively stable in a dry state, but is easily deactivated once dissolved. For example, it is known that BOD is only stable for 96 hours at a limited pH when stored at 5 ° C. in a crude state (Agric. Biol. Chem., 46-82031-2034, 1
982). Several attempts have been reported to stabilize the BOD in order to make the reagent containing the BOD liquid from the time of shipment and use it as it is for measurement.

For example, trishydroxyaminomethane-
An alkaline buffer such as a hydrochloric acid buffer, a stabilizer such as lactose, alanine, glycine, bovine albumin, cyclodextrin, and a stabilizing method containing sodium azide and the like (JP-A-60-151561); A stabilization method containing a specific buffer such as an acid buffer (Japanese Patent Publication No. 62-33880, Japanese Patent Application Laid-Open No. 61-209587)
JP-A-6-28), a stabilizing reagent containing a specific amino acid such as aspartic acid and tryptophan.
No. 4886), a stabilizing method containing a mercapto derivative which is a specific nitrogen-containing heterocyclic compound (JP-A-10-7).
No. 5779) has been proposed.

The present inventor has already proposed a method for stabilizing a BOD in the presence of a reducing agent such as a pentacyanoiron complex for the purpose of stabilizing the BOD in a liquid state for a long period of time (Japanese Patent Laid-Open No. 10-42869). Gazette). On the other hand, the present inventor has proposed a direct bilirubin (conjugated bilirubin) colorimetric method and a reagent for quantitatively determining the combination of BOD with an aniline, a pentacyanoiron complex salt and, if necessary, a cobalt (II) complex as a reaction accelerator. (Japanese Unexamined Patent Publication No. 3-17 / 1991)
No. 5998).

A method for colorimetric determination of bilirubin, characterized in that bilirubin is oxidized by allowing potassium ferricyanide to act on a sample (Japanese Patent Application Laid-Open No. 59-122953).
And BOD mixed with potassium ferricyanide, a reagent for measuring bilirubin (JP-A-59-1980)
No. 30198) has been proposed.

[0008]

However, Japanese Patent Application Laid-Open No. 3-175998 discloses a method for accurately quantifying the amount of bilirubin directly in a sample by accelerating an enzymatic reaction by adding a specific reaction accelerator. But nothing is suggested for the stabilizing effect of BOD.

[0009] JP-A-59-122954 and JP-A-59-130198 only disclose a method for determining bilirubin using potassium ferricyanide and a measuring reagent, and similarly stabilize BOD. No effect is suggested.

[0010] Further, in the above-mentioned Japanese Patent Application Laid-Open No. 10-42869, although the BOD activity can be maintained stably, according to a subsequent study by the present inventors, it was found that when the pentacyanoiron complex salt was used alone. Is a long-term BO
The effect of stabilizing the enzyme activity of D is still insufficient, and a further improved method has been strongly desired.

[0011] Accordingly, the present invention has been made in view of such conventional problems, and a stabilization method capable of stably maintaining the enzyme activity of BOD for a long period of time.
And a reagent composition to which the stabilization technique is applied.

[0012]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that a tertiary amine is further added to a BOD-containing reagent in addition to a pentacyanoiron complex salt and / or a hexacyanoiron complex salt. It has been found that by containing the compound, a high BOD stabilizing effect over a long period of time can be obtained, and the present invention has been achieved.

An object of the present invention is to provide a method for stabilizing a BOD, characterized in that a pentacyanoiron complex salt and / or a hexacyanoiron complex salt and a tertiary amine are contained in a BOD-containing solution, and a method for stabilizing the BOD. This has been achieved by a reagent composition to which the chemical technology is applied.

Hereinafter, the present invention will be described in more detail.

In the present invention, a high stabilizing effect can be achieved by combining a pentacyanoiron complex salt and / or a hexacyanoiron complex salt with a tertiary amine.

The pentacyanoiron complex used in the present invention is:
It can be appropriately selected from known ones and used. Specific examples of the pentacyanoiron complex include compounds represented by the following general formula. M _n [Fe (CN) ₅ X] (where M is an alkali metal or alkaline earth metal, n
Represents an integer of 2 to 5, X is H ₂ O, a halogen atom, S
₍ Indicates ₂ O ₃ , SO ₄ , SCN, CO, NH ₃ , NO ₂ or NO.)

Specific examples of the pentacyanoferrate complex represented by the above general formula include at least one selected from the group consisting of pentacyanoacoferrate (II), pentacyanocarbonylferrate (II) and hydrates thereof. One type is mentioned.

In the present invention, in the BOD-containing solution,
The pentacyanoiron complex salt is added at 2 to 1000 μM, preferably 5 to 1000 μM.
It is contained so as to be 00 μM. When the content of the pentacyanoiron complex salt is less than 2 μM, the stabilizing effect of BOD becomes insufficient. On the contrary, when the content exceeds 1000 μM, the absorbance in the same wavelength region as bilirubin increases, so that the absorbance in the blind test of the reagent increases. Occurs.

The hexacyanoiron complex used in the present invention is also
It can be appropriately selected from known ones and used. Specific examples of the hexacyano iron complex salt include a compound represented by the following general formula. M _n [Fe (CN) ₆ ] (where M is an alkali metal or alkaline earth metal, n
Represents an integer of 2 to 5)

Specific examples of the hexacyanoferrate complex represented by the above general formula include sodium hexacyanoferrate (II), potassium hexacyanoferrate (II), ammonium hexacyanoferrate (II), calcium hexacyanoferrate (II), At least one selected from the group consisting of magnesium hexacyanoiron (II) and hydrates thereof is included.

In the present invention, in the BOD-containing solution,
Hexacyano iron complex is added in an amount of 2 to 1000 μM, preferably 5
It is contained so as to be 00 μM. When the content of the hexacyanoiron complex salt is less than 2 μM, the stabilizing effect of BOD becomes insufficient. On the other hand, when the content exceeds 1000 μM, the absorption is in the same wavelength region as bilirubin like the pentacyanoiron complex salt. The problem that the absorbance becomes high arises.

The tertiary amines used in the present invention can be appropriately selected from known ones regardless of aliphatic amines or aromatic amines. Among these tertiary amines, those having a β-hydroxyalkyl group are particularly preferable, and those having a β-hydroxypropyl group or β-hydroxyethyl group are more preferable.

Examples of such tertiary amines include at least one selected from the group consisting of the compounds listed below or derivatives thereof. N, N-bis (2-hydroxyethyl) glycine (Bici
ne) 2- {4- (2-hydroxyethyl) -1-piperazinyl}
Ethanesulfonic acid (abbreviated as HEPES) N, N-bis (2-hydroxyethyl) -aminoethanesulfonic acid (abbreviated as BES) bis (2-hydroxyethyl) iminotris (hydroxymethyl) ethane (abbreviated as Bis-Tris) 3 -{4- (2-hydroxyethyl) -1-piperazinyl} propanesulfonic acid (abbreviated as EPPS) 2-hydroxy-3- {4- (hydroxyethyl) -piperazinyl} propanesulfonic acid (abbreviated as HEPPSO) 3- ( Morpholino) propanesulfonic acid (abbreviated as MOPS) 2-hydroxy-3-morpholinopropanesulfonic acid
(Abbreviated as MOPS) Piperazine-1,4-bis (ethanesulfonic acid) (PIPE
S) piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (abbreviated as POPSO) 2,2 ′-(n-butylimino) diethanol and tert-butyldiethanolamine

In the present invention, in addition to the pentacyanoiron complex salt and / or hexacyanoiron complex salt, and a tertiary amine, a saccharide having a carboxyl group (hereinafter, referred to as a carboxy sugar) and / or a cobalt (II) complex are contained. By doing so, the stabilizing effect can be further improved, and particularly when a carboxy sugar is contained, the thermal stability can be further improved in addition to the stability at a low temperature.

Specific examples of the carboxy sugar include at least one selected from the group consisting of glucuronic acid, galacturonic acid, mucinic acid and gluconic acid.
Glucuronic acid and mucinic acid are particularly preferred.

This carboxy sugar is 25-1000 m
M, preferably 50 to 700 mM. When the content of this saccharide is less than 25 mM, not only the stabilizing effect becomes insufficient, but also the thermal stability decreases. On the other hand, when the content exceeds 1000 mM, the viscosity of the reagent becomes high, and there is a possibility that the measurement cannot be performed with high accuracy.

Specific examples of the cobalt (II) complex include at least one ligand whose ligand is selected from the group consisting of ethylenediaminetetraacetic acid, CyDTA, diaminopropanoltetraacetic acid, ethylenediaminediacetic acid and diaminopropanetetraacetic acid. Can be

This cobalt (II) complex is 10 to 200
μM, preferably 100 μM.
When the content of the cobalt (II) complex is less than 10 mM,
In some cases, the stabilizing effect is insufficient.

The stabilizing method of the present invention can stably maintain the BOD activity not only in a dry state but also in a liquid state. As the pH of the BOD-containing solution, it is preferable to select a pH effective for maintaining the BOD activity, more preferably pH 5 to 12, and further preferably pH 7.5 to 9.
However, when the storage temperature is high (37 ° C.), pH
It is preferably 8.0 to 12.

A known BOD can be appropriately selected and applied to the stabilizing method of the present invention. Specific examples thereof include, for example, Milosesium Bercaria, Penicillium
BOD derived from an organism selected from the group consisting of Jancinelam, Bacillus rifeniformis, Suehirotake, Asteraceae and alfalfa.

In addition to the BOD derived from these organisms, a recombinant B expressed by recombinantly expressing a gene encoding BOD
Mutant BOD resulting from mutation such as substitution, insertion, or deletion of OD or amino acid sequence is also exemplified as a BOD effective for the stabilization method of the present invention. Further, BODs modified with various carriers with the expectation of stabilizing the enzyme and improving the activity are also included in the BOD according to the present invention. As such modified BODs, those chemically bonded to polyalkylene glycol and the like are known.

In the stabilization method of the present invention, when BOD is used for the measurement of bilirubin, its concentration is generally set to be 0.04 to 10 U / ml in the final reaction solution. More specifically, 0.2-10 U / ml for total bilirubin measurement and 0.1-10 U / ml for direct bilirubin measurement.
About -5 U / ml is preferable.

In the present invention, a BOD-containing reagent composition having an excellent stabilizing effect by applying the above stabilizing technique can be provided. The reagent composition contains pentacyanoiron complex and / or hexacyanoiron complex, tertiary amines and BOD at the required concentrations. The pentacyano iron complex salt, hexacyano iron complex salt, and tertiary amines contained in this reagent composition can be appropriately selected from those described above and used.

The reagent composition of the present invention further contains a carboxy sugar and / or a cobalt (II) complex in addition to the pentacyanoiron complex salt, hexacyanoiron complex salt, and tertiary amine to further enhance the stabilizing effect. Can be improved, especially when a carboxy sugar is contained,
In addition to stability at low temperatures, thermal stability can be further improved.

The reagent composition of the present invention is not particularly required, but may be added with known stabilizers such as saccharides, amino acids, and inactive proteins as necessary.
OD stabilization can be further enhanced. Examples of the saccharide include lactose and saccharose, examples of the amino acid include aspartic acid and tryptophan, and examples of the inactive protein include serum albumin and ovalbumin of bovine, goat, sheep, and horse.

Further, a surfactant or the like may be added to the reagent composition of the present invention to promote the reaction when measuring total virbilin. As the surfactant, an anionic surfactant such as sodium cholate or sodium lauryl sulfate is preferably used.

[0037]

According to the present invention, the enzyme activity of BOD can be stably maintained over a long period of time not only in a dry state but also in a solution state. Therefore, according to the present invention, bilirubin in a serum sample can be measured with high accuracy, so that liver function and hematopoietic function-related diseases can be diagnosed with high accuracy. Further, in the present invention, since the enzyme activity of BOD can be maintained at a high level, it is not necessary to use an excess enzyme to compensate for the decrease in the activity of the enzyme.

[0038]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.

Test Example 1 Measurement of BOD enzyme activity 30 mg albumin-conjugated bilirubin (manufactured by Amano Pharmaceutical Co., Ltd.)
With 0.05M phosphate buffer (pH 7.0, EDTA)
(Contained) and dissolved in 1 mL to obtain a substrate solution. 0.5 mg of sodium cholate (manufactured by Nacalai Tesque) was dissolved in 50 mL of the same phosphate buffer to prepare a buffer containing sodium cholate. After 0.2 mL of the substrate solution and 3 mL of the buffer solution containing sodium cholate were added to the quartz cell and mixed well, and allowed to stand for 10 minutes, 0.1 mL of a BOD (Mirothecium-derived microorganism-derived BOD: manufactured by Amano Pharmaceutical) was added. After mixing and reacting at 37 ° C., a spectrophotometer (manufactured by Hitachi, Ltd .:
The change rate of the absorbance at 1 minute and 3 minutes after the start of the reaction was measured at a wavelength of 460 nm using a 705 type Hitachi automatic analyzer.
As a control, the same operation was performed by adding a phosphate buffer instead of the substrate solution, and the absorbances Ab1 (after one minute) and Ab3 (3
Minutes later) were measured. Under these conditions, the amount of enzyme (1 unit) required to oxidize 1 μmol of albumin-bound bilirubin per minute was calculated by the following equation.

[0040]

(Equation 1)

Embodiment 1 Pentacyanoiron complex and tertiary iron
BOD Stabilizing Effect by Combination of Tertiary Amines To the following BOD-containing composition solution, 20 mM of each of the following (1) was added as a tertiary amine and (2) to (5) were added, and 14, 21, 29 days, 4,
Except for storage at 10, 25, and 37 ° C., the enzyme activity was measured in exactly the same manner as in Test Example 1, and the stabilizing effect of BOD was examined. BOD-containing composition solution TAPS-NaOH buffer 20.0 mM EDTA · 2Na 1.00 mM BOD 2.0 U / ml Na ₃ [Fe (CN) ₅ (H ₂ O)] 10 μM pH 8.50 Tertiary amines (1 ) No addition (2) POPSO (3) HEPSO (4) EPPS (5) Bicine

Table 1 shows the results. As shown in Table 1, when the pentacyano iron complex salt and the tertiary amines are combined {(2) to (5)}, the stabilizing effect is remarkably higher as compared with the case of using the pentacyano iron complex salt alone (1). You can see that it has improved. In particular, in the cases of (2) to (4), it can be seen that the stabilizing effect is high and the thermal stability is excellent.

[0043]

[Table 1]

Comparative Example 1 BO with tertiary amines alone
Example 1 Stabilizing effect of D Example 1 except that no pentacyanoiron complex salt was added.
The enzymatic activity was measured in exactly the same manner as described above, and the stabilizing effect of BOD was examined. Table 2 shows the results. As shown in Table 2, it can be seen that the tertiary amine alone does not provide an effective stabilizing effect. This shows that, in consideration of Example 1, a synergistic effect was produced only when the pentacyanoiron complex salt and the tertiary amine were combined.

[0045]

[Table 2]

Embodiment 2 FIG. BOD stabilizing effect of tertiary amines produced when combined with pentacyanoiron complex salt The following tertiary amines (1)
No addition, 20 mM of each of (2) to (13) was added,
0, 3, 7, 14, 22, 35, 42, 56, 70 days at 4, 10 ° C. respectively, 0, 3, 14, 2
Store at 25 ° C. for 2, 35, 42, 56 days, 0, 3,
The enzymatic activity was measured in exactly the same manner as in Example 1 except that it was stored at 37 ° C. for 7, 14, and 22 days, and the stabilizing effect of BOD was examined. BOD-containing composition solution TAPS-NaOH buffer 20.0 mM EDTA · 2Na 1.00 mM BOD 2.0 U / mL Na ₃ [Fe (CN) ₅ (H ₂ O)] 100.0 μM pH 8.50 Tertiary amines (1) No addition (2) Bicine (3) HEPES (4) BES (5) Bis-Tris (6) EPPS (7) HEPPSO (8) MOPS (9) MOPSO (10) PIPES (11) POPSO (12) 2,2 '-(n-butylimino) diethanol (13) tert-butyldiethanolamine

Table 3 shows the results. As shown in Table 3, when tertiary amines were added, no addition (1)
It can be seen that the effect of stabilizing the BOD is superior to the case of the above. In particular, tertiary amines (2) to (7) and (11)
When (13) to (13) are added, the stabilizing effect is further improved, and the tertiary amines (1), (4) to (6) and (1)
It can be seen that when 0) was added, the stabilizing effect was further improved, and when the tertiary amines (6) and (10) were added, the stabilizing effect was further improved. This suggests that those containing a β-hydroxy group can achieve a high stabilizing effect, and that those containing a β-hydroxyethyl group can achieve a higher stabilizing effect.

[0048]

[Table 3]

Embodiment 3 FIG. BOD stabilizing effect by combination of pentacyano iron complex salt, tertiary amines and carboxy sugar In the BOD-containing composition solution used in Example 2, 20 mM tertiary amines (1) were used as carboxy sugars (A) No addition and (B) added to 100 mM glucuronic acid, respectively, and 20 mM tertiary amines (2) were added as carboxy sugars to (A) no addition and (B) 100 mM glucuronic acid, respectively, as carboxy sugars. Tertiary amines (3) were added as carboxy sugars without (A), (B) 1
00 mM glucuronic acid, (C) 100 mM galacturonic acid, (D) 100 mM sodium gluconate,
(E) added to 100 mM mucinic acid, respectively;
Stored at 4, 10 ° C. for 3, 7, 14, 22, 35, 42, 56, 70 days, respectively, 0, 3, 7, 14, 22,
Stored at 25 ° C. for 35, 42, 56 days, 0, 3, 7,
The enzymatic activity was measured in exactly the same manner as in Example 1 except for storing at 37 ° C. for 14, 22 days, and the stabilizing effect of BOD was examined. Tertiary amines (1) Bicine (2) HEPES (3) POPSO Carboxy sugar (A) No addition (B) Glucuronic acid (C) α-D-galacturonic acid (D) Sodium gluconate (E) Mucinic acid

Table 4 shows the results. As shown in Table 4, when the pentacyano iron complex salt, the tertiary amines and the carboxy sugar were combined, compared with the case where the pentacyano iron complex salt and the tertiary amines were combined (no addition: Example 2) ) It can be seen that the stabilizing effect was further improved. In particular, it can be seen that the thermal stability was significantly improved during high-temperature storage (37 ° C.) as compared with the case where no additive was added.

[0051]

[Table 4]

Comparative Example 2 Stabilizing effect of BOD by carboxy sugar alone (without adding tertiary amines) Enzyme activity was measured in exactly the same manner as in Example 3 except that tertiary amines were not added, and stabilizing effect of BOD Was examined. Table 5 shows the results. As shown in Table 5, when tertiary amines are not added, not only no stabilizing effect is obtained, but also a case where they are destabilized is seen. This means that the carboxy sugar, when combined with the pentacyanoiron complex, has a stabilizing effect that is not as great as that of the pentacyanoiron complex alone, but only when combined with the pentacyanoiron complex and the tertiary amine. This indicates that the effect has improved.

[0053]

[Table 5]

Embodiment 4 FIG. B by combination of pentacyanoiron complex, tertiary amines and cobalt (II) complex
OD stabilizing effect In the BOD-containing composition solution used in Example 2, 20 mM tertiary amines (POPSO, HEPES) were not added as carboxy sugars (1) without addition and (2) 100 mM sodium gluconate and cobalt ( II) As a complex (1)
No addition and (2) 100 μM Co (II) -EDTA
, And stored at 4, 10, 25, and 37 ° C. for 0, 3, 7, 14, 21, and 35 days, respectively.
The stabilizing effect of D was investigated.

Table 6 shows the results. As shown in Table 6, the cobalt (II) complex has a more stabilizing effect when combined with a pentacyanoiron complex, a tertiary amine and a carboxy sugar than when combined with a pentacyanoiron complex and a tertiary amine. Is improved, and a higher temperature (3
7 ° C.), the thermal stability is improved.

[0056]

[Table 6]

Comparative Example 3 B without tertiary amines
OD stabilizing effect Except that tertiary amines were not added, the enzyme activity was measured in exactly the same manner as in Example 4, and the BOD stabilizing effect was examined. Table 7 shows the results. As shown in Table 7, when the tertiary amines were not added, no stabilizing effect was obtained regardless of the presence or absence of the carboxy sugar. This means that the cobalt (II) complex, when combined with a pentacyano iron complex salt and a tertiary amine, has an improved stabilizing effect for the first time, and further, when combined with a pentacyano iron complex salt, a tertiary amine and a carboxy sugar. In some cases, the stabilizing effect is improved.

[0058]

[Table 7]

Embodiment 5 FIG. Stabilizing effect of pentacyano iron complex salt and hexacyano iron complex salt The following pentacyano iron complex salts (1) to (3) and (5) to (6) or hexacyano iron complex salt (4) were added to the following BOD-containing composition solution. 100 mM added, 0,
Enzyme activity was measured in exactly the same manner as in Example 1 except that it was stored at 4, 10, and 25 ° C. for 3, 7, 14, 21, 28, 42, and 70 days, respectively, and the stabilizing effect of BOD was examined. .

Table 8 shows the results. As shown in Table 8, a significantly higher stabilizing effect can be obtained as compared with the case where neither the pentacyanoiron complex salt nor the hexacyanoiron complex salt is added (containing tertiary amines, cobalt complex and carboxy sugar). I understand. BOD-containing composition solution Tris buffer 50.0 mM EDTA · 2Na 1.00 mM POPSO 20 mM BOD 2.0 U / ml Co (II) -EDTA 100.0 μM D-glucuronic acid 100.0 mM pH 8.50 Pentacyan iron complex and hexacyano Iron complex salts (1
(00 μM) (1) Na ₃ [Fe (CN) ₅ (H ₂ O)] (2) Na ₃ [Fe (CN) ₅ Cl] (3) Na ₃ [Fe (CN) ₅ Br] (4) Na ₄ [Fe (CN) ₆ ] (5) Na ₅ [Fe (CN) ₅ (SCN)] (6) Na ₅ [Fe (CN) ₅ (S ₂ O ₅ )]

[Table 8]

Embodiment 6 FIG. Effect of stabilizing BOD under the condition that the same storage container is not used (opening-sealing-opening-sealing is not repeated) Enzyme activity exactly as in Example 5, except that the same storage container was not used Was measured, and the stabilizing effect of BOD was examined. Table 9 shows the results. As shown in Table 9, when the same storage container was not used, the stabilizing effect was remarkably higher than when the same storage container of Example 5 was used (repeating conditions from opening to sealing to opening to sealing). You can see that it has improved. This is considered to be due to the condition that dissolved oxygen, which is a cause of destabilizing the BOD, is not supplied.

[0062]

[Table 9]

Example 7 Stabilizing Effect in the Concentration Range of Pentacyanoiron Complex Salts and Hexacyanoiron Complex Salts The following pentacyanoiron complex salts (1) to (2) and hexacyanoiron complex solution were added to the same BOD-containing composition solution as in Example 5 above. Complex salts (3) were prepared at 5, 10, 50, 100 and 50, respectively.
0, 3, 7, 14, 21, 28, 4
Enzyme activity was measured in exactly the same manner as in Example 1 except that the samples were stored at 4, 10, and 25 ° C. for 2, 70 days, respectively.
The stabilizing effect of OD was examined. Pentacyano iron complex salts (1) Na ₃ [Fe (CN) ₅ (H ₂ O)] (2) Na ₃ [Fe (CN) ₅ Br] hexacyano iron complex salts (3) Na ₄ [Fe (CN) ₆ ]

Tables 10 to 12 show the results. Table 10
~ As shown in Table 12, pentacyano iron complex salt (1) ~
Both (2) and hexacyanoiron complex salt (3) have a higher stabilizing effect than those in the case where they are not added (containing tertiary amines, cobalt complex and carboxy sugar). It can be seen that a higher stabilizing effect can be obtained.

[Table 10]

[0065]

[Table 11]

[0066]

[Table 12]

Embodiment 8 FIG. Stabilizing effect in the concentration range of tertiary amines The following tertiary amines (1)
To (3) are 0.1, 1, 10, 20, 50, 1
00, 500 mM, 0, 3, 7, 14, 21, 2
The enzymatic activity was measured in exactly the same manner as in Example 1 except that they were stored at 4, 10, and 25 ° C. for 8, 42, and 70 days, respectively, and the stabilizing effect of BOD was examined. BOD-containing composition solution Tris buffer 50.0 mM EDTA · 2Na 1.00 mM Na ₃ [Fe (CN) ₅ (H ₂ O)] 100 μM BOD 2.0 U / ml Co (II) -EDTA 100.0 μM D-glucuronic acid 100.0 mM pH 8.50 Tertiary amines (1) EPPS (2) HEPPSO (3) POPSO

Tables 13 to 15 show the results. Table 13
As shown in Table 15, in all the tertiary amines, a higher stabilizing effect can be obtained by adding 1 mM or more than in the case where no tertiary amine is added. It can be seen that a high stabilizing effect can be obtained. Especially H
In the case of EPPSO and POPSO, a high stabilizing effect can be obtained even with the addition of 0.1 mM.

[0069]

[Table 13]

[0070]

[Table 14]

[0071]

[Table 15]

Embodiment 9 FIG. Stabilizing effect in carboxy sugar concentration range The following carboxy sugar (1) was added to the following BOD-containing composition solution.
1, 10, 25, 50, 100, 200, and 500 mM were added, and the following carboxy sugars (2) to (4) were added in 1
0, 25, 50, 100, 200, 500 mM added,
The enzymatic activity was measured in exactly the same manner as in Example 1 except that they were stored at 4, 10 ° C. for 0, 7, 14, 22, 35, 42, and 70 days, respectively, and the stabilizing effect of BOD was examined.

Tables 16 to 19 show the results. Table 16
As shown in Table 19, in any of the carboxy sugars, a higher stabilizing effect was obtained by adding 25 mM or more than in the case of no addition, and the higher the concentration, the higher the stabilization effect. It turns out that an effect is obtained. In particular, in the case of glucuronic acid and α-D-galacturonic acid, 2
A high stabilizing effect can be obtained even at 10 ° C. by adding 00 mM or more. BOD-containing composition solution Tris buffer 50.0 mM EDTA · 2Na 1.00 mM Na ₃ [Fe (CN) ₅ (H ₂ O)] 100.0 μM BOD 2.0 U / ml Co (II) -EDTA 100.0 μM POPSO 100 0.0 mM pH 8.50 Carboxy sugar (1) Glucuronic acid (2) α-D-galacturonic acid (3) Sodium gluconate (4) Mucinic acid

[0074]

[Table 16]

[0075]

[Table 17]

[0076]

[Table 18]

[0077]

[Table 19]

Embodiment 10 FIG. Cobalt complex @ Co (II) -E
Stabilizing effect in the concentration range of DTA} 20 mM POPSO or HEPES was added to the following BOD-containing composition solution, and 0, 5, 25, 50, 10
0, 250 μM Co (II) -EDTA were added respectively, and 0, 3, 7, 14, 21, 35 days, 4,
Stored at 10, 25 ° C for 0, 3, 7, 14, 21 days,
Except for storage at 37 ° C., the enzymatic activity was measured in exactly the same manner as in Example 1, and the stabilizing effect of BOD was examined.

The results are shown in Tables 20 and 21. Table 20
As shown in Table 21, regardless of whether POPSO or HEPES is used, a high stabilizing effect can be obtained particularly at high temperatures by adding Co (II) -EDTA.
In addition, it is understood that a higher stabilizing effect can be obtained when POPSO is used than HEPES. BOD-containing composition solution Tris buffer 50.0 mM EDTA · 2Na 1.00 mM Na ₃ [Fe (CN) ₅ (H ₂ O)] 100.0 μM BOD 2.0 U / ml Glucuronic acid 100.0 mM POPSO or HEPES 20.0 mM pH 8.50

[0080]

[Table 20]

[0081]

[Table 21]

Embodiment 11 FIG. Stabilizing effect in pH range 20 mM H as a buffer was added to the following BOD-containing composition solution.
Add EPES, CHES and CAPS, pH7.
0, 3, 7, 14, 21, 28 in the range of 0 to 11.0,
Enzyme activity was measured in exactly the same manner as in Example 1 except that the samples were stored at 4, 10, 25, and 37 ° C. for 35 days, respectively.
The stabilizing effect of BOD was examined.

Table 22 shows the results. As shown in Table 22, a particularly high stabilizing effect is obtained in the pH range of 7.5 to 9.0, and when the storage temperature is high (37 ° C.), p
It can be seen that the higher the H, the higher the stability. BOD-containing composition solution EDTA · 2Na 1.00 mM BOD 2.0 U / ml Co (II) -EDTA 100.0 μM D-glucuronic acid 100.0 mM

[0084]

[Table 22]

Claims (26)

[Claims] 1. A pentacyanoiron complex and / or hexacyanoiron complex in a solution containing bilirubin oxidase.
And a method for stabilizing bilirubin oxidase, comprising a tertiary amine. 2. The content of the pentacyanoiron complex salt is 2 to 100.
The method for stabilizing bilirubin oxidase according to claim 1, wherein the concentration is in the range of 0 µM. 3. The method for stabilizing bilirubin oxidase according to claim 1, wherein the pentacyanoiron complex salt is a compound represented by the following general formula. M _n [Fe (CN) ₅ X] (where M is an alkali metal or alkaline earth metal, n
Represents an integer of 2 to 5, X is H ₂ O, a halogen atom, S
₍ Indicates ₂ O ₃ , SO ₄ , SCN, CO, NH ₃ , NO ₂ or NO.) 4. The pentacyano iron complex salt of pentacyano iron
The method for stabilizing bilirubin oxidase according to any one of claims 1 to 3, wherein the method is at least one selected from the group consisting of (II) acid salt, pentacyanocarbonyl iron (II) acid salt and a hydrate thereof. 5. The content of the hexacyanoiron complex salt is 2 to 100.
The method for stabilizing bilirubin oxidase according to claim 1, wherein the concentration is in the range of 0 µM. 6. The method for stabilizing bilirubin oxidase according to claim 1, wherein the hexacyanoiron complex salt is a compound represented by the following general formula. M _n [Fe (CN) ₆ ] (where M is an alkali metal or alkaline earth metal, n
Represents an integer of 2 to 5) 7. The method according to claim 1, wherein the hexacyanoiron complex salt is hexacyanoiron (I
I) sodium acid, potassium hexacyanoferrate (II),
6. The method for stabilizing bilirubin oxidase according to claim 5, wherein the method is at least one selected from the group consisting of ammonium hexacyanoferrate (II), calcium hexacyanoferrate (II), magnesium hexacyanoferrate (II), and a hydrate thereof. . 8. A tertiary amine having a content of 0.01 to 10%.
The method for stabilizing bilirubin oxidase according to claim 1, wherein the amount is in the range of 00 mM. 9. The method for stabilizing bilirubin oxidase according to claim 7, wherein the tertiary amine is at least one selected from the group consisting of the compounds listed below and derivatives thereof. N, N-bis (2-hydroxyethyl) glycine 2- [4- (2-hydroxyethyl) -1-piperazinyl]
Ethanesulfonic acid N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid bis (2-hydroxyethyl) -iminotris (hydroxymethyl) ethane 3- [4- (2-hydroxyethyl) -1-piperazinyl ]
Propanesulfonic acid 2-hydroxy-3- [4- (hydroxyethyl) -1-
Pyrazinyl] propanesulfonic acid 3-morpholinopropanesulfonic acid 2-hydroxy-3-morpholinopropanesulfonic acid piperazine-1,4-bis (ethanesulfonic acid) piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid ) 2,2 '-(n-butylimino) diethanol and tert-butyldiethanolamine 10. The method for stabilizing bilirubin oxidase according to claim 1, further comprising a saccharide having a carboxyl group. 11. The method for stabilizing bilirubin oxidase according to claim 10, wherein the content of the saccharide having a carboxyl group is in the range of 25 to 1000 mM. 12. The saccharide having a carboxyl group is at least one selected from the group consisting of glucuronic acid, galacturonic acid, gluconic acid and mucinic acid.
Or the method for stabilizing bilirubin oxidase according to 11 above. 13. The method for stabilizing bilirubin oxidase according to claim 1, further comprising a cobalt (II) complex. 14. The ligand of the cobalt (II) complex is at least one selected from the group consisting of ethylenediaminetetraacetic acid, CyDTA, diaminopropanoltetraacetic acid, ethylenediaminediacetic acid and diaminopropanetetraacetic acid. Of stabilizing bilirubin oxidase. 15. The content of the cobalt (II) complex is 2-10.
The method for stabilizing bilirubin oxidase according to claim 13 or 14, wherein the amount is in the range of 00 µM. 16. The method for stabilizing bilirubin oxidase according to claim 1, wherein the pH of the solution is in the range of 5.0 to 12.0. 17. A reagent composition comprising a pentacyano iron complex salt and / or a hexacyano iron complex salt, a tertiary amine, and a virbilin oxidase. 18. The reagent composition according to claim 17, wherein the pentacyanoiron complex salt is a compound represented by the following general formula. M _n [Fe (CN) ₅ X] (where M is an alkali metal or alkaline earth metal, n
Represents an integer of 2 to 5, X is H ₂ O, a halogen atom, S
₍ Indicates ₂ O ₃ , SO ₄ , SCN, CO, NH ₃ , NO ₂ or NO.) 19. The pentacyanoferrate complex salt is at least one selected from the group consisting of pentacyanoacoferrate (II), pentacyanocarbonylferrate (II), and hydrates thereof.
19. The reagent composition according to claim 17 or 18, which is a species. 20. The reagent composition according to claim 17, wherein the hexacyanoiron complex salt is a compound represented by the following general formula. M _n [Fe (CN) ₆ ] (where M is an alkali metal or alkaline earth metal, n
Represents an integer of 2 to 5) 21. The hexacyanoiron (II) complex salt is sodium hexacyanoferrate (II), potassium hexacyanoferrate (II), ammonium hexacyanoferrate (II), calcium hexacyanoferrate (II), magnesium hexacyanoferrate (II), and magnesium hexacyanoferrate (II). 21. The reagent composition according to claim 20, which is at least one selected from the group consisting of hydrated salts. 22. The reagent composition according to claim 17, wherein the tertiary amine is at least one selected from the group consisting of the compounds listed below or derivatives thereof. N, N-bis (2-hydroxyethyl) glycine 2- [4- (2-hydroxyethyl) -1-piperazinyl]
Ethanesulfonic acid N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid bis (2-hydroxyethyl) -iminotris (hydroxymethyl) ethane 3- [4- (2-hydroxyethyl) -1-piperazinyl ]
Propanesulfonic acid 2-hydroxy-3- [4- (hydroxyethyl) -1-
Pyrazinyl] propanesulfonic acid 3-morpholinopropanesulfonic acid 2-hydroxy-3-morpholinopropanesulfonic acid piperazine-1,4-bis (ethanesulfonic acid) piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid ) 2,2 '-(n-butylimino) diethanol tert-butyldiethanolamine 23. The reagent composition according to claim 17, further comprising a saccharide having a carboxyl group. 24. The reagent composition according to claim 23, wherein the saccharide having a carboxyl group is at least one selected from the group consisting of glucuronic acid, galacturonic acid and gluconic acid. 25. The method according to claim 1, which comprises a cobalt (II) complex.
25. The reagent composition according to 7 to 24. 26. The ligand of the cobalt (II) complex is at least one selected from the group consisting of ethylenediaminetetraacetic acid, CyDTA, diaminopropanoltetraacetic acid, ethylenediaminediacetic acid and diaminopropanetetraacetic acid. Reagent composition.