JPH1160600A - Stabilization of myoglobin in urine, stabilization agent and immunoassay using the agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stabilize myoglobin in urine, preventing the lowering of the immunologically measured value of myoglobin in urine even after freezing and thawing treatment and useful for the diagnosis, etc., of cardiac diseases, renal diseases, etc., by adding a bicarbonate compound to a urine specimen. SOLUTION: The myoglobin level in urine is stabilized to prevent the lowering of immunologically measured value of myoglobin in urine not only in the case of low-temperature storage but also after the freezing and thawing treatment by incorporating a urine specimen with a bicarbonate compound such as sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate in an amount of 6.25-200 mM and further with 3.9-500 mM of an azide such as sodium azide, potassium azide and ammonium azide, 5-160 mM of a chelating agent such as EDTA (salt) and 0.01-0.2 w/v% of a nonionic surfactant such as a sorbitan fatty acid ester and/or a polyoxyethylene sorbitan fatty acid ester.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing urinary myoglobin, and more particularly to a method for stabilizing urinary myoglobin not only when it is stored at low temperatures but also when it is frozen and thawed. The present invention relates to a method for stabilizing urinary myoglobin which can prevent the occurrence of myoglobin.

[0002]

2. Description of the Related Art Myoglobin is a heme protein present in animal skeletal muscle tissue and plays a role in storing oxygen. Human myoglobin has a molecular weight of about 17,500, and has a structure in which one heme molecule is added to one peptide chain composed of 153 amino acid residues. When muscle tissue damage such as myocardial infarction occurs, blood and urine levels of myoglobin significantly increase within a few hours. in recent years,
It has also been pointed out that an increase in myoglobin concentration associated with muscle tissue damage leads to kidney damage (Clin. Nephrol., 38, 1
93-5; 1992).

[0003] Therefore, measuring the myoglobin concentration in urine with high precision is extremely important in diagnosing heart disease and kidney disease.

Conventionally, urinary myoglobin has been measured using a urine test strip. However, this method is based on a reaction based on the peroxidase-like activity of myoglobin as an index, so that hemoglobin having the same catalytic activity as that of hemoglobin has a similar catalytic activity. Interference cannot be avoided. Therefore, when performing this method, an operation for separating hemoglobin in advance has been required.

On the other hand, for serum samples, a method for immunologically measuring myoglobin is known (Clin. Chem., 36, 16).
75-8; 1990) (Ann. Clin. Biochem., 28, 474-9; 1991) (Cl
in. Chim. Acta., 209, 197-207; 1992). If an antibody specific to human myoglobin is used, there is no interference from hemoglobin, so that myoglobin can be specifically measured without performing complicated separation operations.

However, when the method for measuring a serum sample is applied to a urine sample, the stability of myoglobin in the urine sample becomes a problem. Generally, myoglobin in serum can be kept to a negligible level by measuring the fluctuation of serum by freezing or refrigerated storage of serum. However, in the case of myoglobin in urine, there is a sample in which the measured value of myoglobin is greatly reduced even if urine is frozen immediately after collection. In particular, when the pH of urine is low, it is known that the measured value of myoglobin significantly decreases.

[0007] It has been found that the storage stability of urinary myoglobin can be improved by adjusting the pH to 8.0 to 9.5, and there is a report applied to immunological measurement (Cli).
n. Chem., 40, 796-802; 1994). In this report, the pH of urine samples was adjusted to alkaline with NaOH,
It has been confirmed that stable measurement values can be obtained over two days.

However, subsequent studies by the present inventors have revealed that the method of making a urine sample alkaline is not necessarily effective for all urine samples. That is, although the measured value of myoglobin can be maintained stably to some extent in some samples, many samples in which no stabilizing effect can be expected were observed. In addition, when the pH was adjusted with an alkaline component such as NaOH, a specimen that caused precipitation was often observed. Urine sedimentation is not a desirable phenomenon because it interferes with the collection of a uniform sample.

Furthermore, when a urine sample was frozen, a phenomenon was observed in which the measured value of myoglobin was reduced each time the freeze-thaw operation was performed. The phenomenon of decrease in measured values due to freezing and thawing could not be eliminated by the alkali treatment described above.

On the other hand, a technique has been proposed in which a urine sample is preliminarily alkali-treated and then used as a sample for immunological measurement (Japanese Patent Application Laid-Open No. 8-166382). In doing so, an alkaline component is used to suppress the effects of components that inhibit the reaction.

Further, a technique has been proposed in which a bicarbonate salt of about 50 mM is added to urine to prevent the phenomenon of clouding under cold conditions (Japanese Patent Application Laid-Open No. 53-141695). It has been further improved.

However, the above publications merely disclose techniques for preventing a phenomenon that inhibits the reaction when measuring a certain analyte component in a urine sample. No gender is suggested.

For the purpose of improving the storage stability of this urinary myoglobin, a stabilizer comprising a predetermined amount of sodium azide and EDTA / 3Na has been reported (Medical and Pharmacy, 37, 1255-9). ; 1997).

[0014]

However, this stabilizer can stabilize myoglobin in urine for 3 weeks when stored at 4 ° C. According to the results, it was confirmed that the measured values significantly decreased due to freeze-thaw.

Accordingly, the present invention has been made in view of such conventional problems, and is effective not only when stored at low temperature but also when frozen and thawed for a long period of time. Method to stabilize urinary myoglobin,
An object of the present invention is to provide a stabilizer and a method for immunologically measuring urinary myoglobin using the same.

[0016]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, the urine sample was stored at a low temperature by containing a bicarbonate compound and, if necessary, an azide. In addition to the case, the present inventors have found that myoglobin in urine can be stabilized for a long period of time, even in the case of freeze-thawing, and have reached the present invention.

An object of the present invention is to provide a method for stabilizing urinary myoglobin, characterized in that a bicarbonate compound and, if necessary, an azide are contained in a urine sample, and a stabilizing agent and application thereof. Achieved by the immunoassay method described.

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

The bicarbonate compound used in the present invention can be appropriately selected from known bicarbonate compounds and used. Specific examples include at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate, and ammonium bicarbonate. In the method for stabilizing urinary myoglobin of the present invention, when a urine sample contains sodium bicarbonate, it becomes alkaline, and when it contains ammonium bicarbonate, it becomes neutral.

In the present invention, a bicarbonate compound is contained in a urine sample in an amount of 6.25 to 200 mM, preferably 12.5 to 1 mM.
It is contained so as to be 00 mM, more preferably 100 mM. When the content of the bicarbonate compound is less than 6.25 mM, the stabilizing effect may be insufficient depending on the urine sample. On the other hand, when the content exceeds 200 mM, precipitation tends to occur, and the measured value is rather lowered.

In the present invention, the stabilizing effect of urinary myoglobin can be further improved by adding an azide in addition to the bicarbonate compound. Specific examples thereof include, for example, sodium azide,
At least one selected from the group consisting of potassium azide and ammonium azide is included.

This azide is present in the urine sample from 3.9 to 50
0 mM, preferably 7.8-250 mM, more preferably 6 mM
It is contained so as to be 2.5 mM. If the azide content is less than 3.9 mM, the stabilizing effect becomes insufficient, and if it exceeds 500 mM, the stabilizing effect cannot be further improved.

As mentioned above, the addition of a bicarbonate compound to a urine sample may cause precipitation.
In addition, this precipitate is also generated due to factors such as the type and concentration of salts contained in urine and pH. This precipitation can be prevented by including a chelating agent in the urine sample, and the stabilizing effect of urinary myoglobin can be further improved. However, since this precipitate is not always generated due to the addition of the bicarbonate compound and the salts and pH in urine, a chelating agent may be added as needed.

The chelating agent is contained in the urine sample at 1.25 to 16
0 mM, preferably 5 to 80 mM, more preferably 40 mM. The content of the chelating agent is 1.25
When the amount is less than mM, precipitation occurring in urine cannot be effectively prevented, so that the stabilizing effect becomes insufficient. On the other hand, when the amount exceeds 160 mM, the immune reaction is inhibited, and the measured value decreases.

The chelating agent having an action of preventing precipitation used in the present invention can be appropriately selected from known chelating agents and used. Such chelating agents include:
For example, ethylenediaminetetraacetic acid (EDTA), 2-cyclohexanediaminetetraacetic acid, methylenediaminetetraacetic acid, iminodiacetic acid and the like can be mentioned, and EDTA or a salt thereof is particularly preferable.

In the present invention, by adding an azide and a chelating agent to the above-mentioned bicarbonate compound as required, a sufficient stabilizing effect can be obtained not only in the case of low-temperature storage but also in the case of freeze-thawing. Although it can be obtained, the measurement value accompanying freeze-thaw may decrease depending on the sample. In this case, by adding a surfactant in addition to the bicarbonate compound, the azide and the chelating agent, a high stabilizing effect can be maintained even in the case of freeze-thawing.

The surfactant used in the present invention can be appropriately selected from known ones. Among these surfactants, nonionic surfactants are particularly preferred, and sorbitan fatty acid esters and / or
Or a polyoxyethylene sorbitan fatty acid ester, more preferably a polyoxyethylene sorbitan fatty acid ester.

Specific examples of the polyoxyethylene sorbitan fatty acid ester include at least one selected from the group consisting of the compounds listed below and derivatives thereof. Polyoxyethylene sorbitan monolaurate [trade name: Tween 20 manufactured by Kao Atlas Co., Ltd.] Polyoxyethylene sorbitan monopalmitate [trade name: Tween 40 manufactured by Kao Atlas Co., Ltd.] Polyoxyethylene sorbitan monostearate [Kao Atlas (Trade name: Tween 60) Polyoxyethylene sorbitan tristearate (trade name, manufactured by Kao Atlas Co., Ltd .: Tween 65) Polyoxyethylene sorbitan monooleate (trade name, manufactured by Kao Atlas Co., Ltd .: Tween 80) Polyoxyethylene sorbitan trioleate [Trade name: Tween 85, manufactured by Kao Atlas Co., Ltd.]

This surfactant is used in an amount of 0.01 to 0.2 w / v.
%, Preferably 0.025 to 0.1 w / v%. If the content of the surfactant is less than 0.01 w / v%, the stabilizing effect accompanying freeze-thawing may be insufficient, whereas if it exceeds 0.2 w / v%, the immune reaction is inhibited, On the contrary, the measured value decreases.

In the present invention, although not particularly required, the stabilizing effect of myoglobin can be further enhanced by adding a known protein protective agent as needed. Examples of such a protein protecting agent include inactive proteins represented by albumin and gelatin.

Any albumin can be used, but albumin derived from animal serum or eggs is particularly preferred. Specific examples include cows, horses, goats,
Sheep, pigs, rabbits, and albumins derived from fetal blood or fetal blood of these animals. In addition to those described above, enzymatically degraded products of this albumin are also known, but albumin in the present invention may include proteins derived from such albumin.

The present invention can provide a urinary myoglobin stabilizer containing a bicarbonate compound acting as a urinary myoglobin stabilizing factor.
In this case, the bicarbonate compound to be added can be appropriately selected from at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate and ammonium bicarbonate.

Further, the stabilizer of the present invention may contain an azide, a chelating agent and a surfactant, if necessary. The azide, chelating agent and surfactant can be appropriately selected from those described above and used.

The urine sample in which myoglobin has been stabilized according to the present invention is directly used as a sample for measuring myoglobin concentration based on a known immunological measurement method. Specific examples of the measurement method include RIA based on the principles of the sandwich method and the competition method.
Method, an ELISA method, an immunological particle agglutination method such as a latex agglutination method, and an immunoturbidimetric method.

The urine sample is appropriately diluted according to the measurable range of the reagent to be used to prepare a sample for measurement. For dilution,
A diluting solution that provides a suitable pH or salt concentration for an immunological reaction may be used.

[0036]

According to the present invention, the immunological measurement value of urinary myoglobin can be stably maintained over a long period of time, not only when it is stored at low temperature but also when it is frozen and thawed.
Therefore, according to the immunological measurement method of the present invention, a heart disease or a kidney disease can be diagnosed with high accuracy.

[0037]

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.

Example 1 0, 6.25, 12.5, 25, 50, 10
Add 0, 200, 400 mM sodium bicarbonate and add 4
The samples were stored at 0 ° C for 0, 1, 4, and 7 days, respectively, to examine the effect of the myoglobin on immunological measurements.

For the immunological measurement, a commercially available kit for measuring myoglobin IRMA, "Ab beads myoglobin 'Eiken'" (trade name, manufactured by Eiken Chemical Co., Ltd.) was used. This kit is for performing IRMA based on a solid phase sandwich method using a ¹²⁵ I-labeled anti-myoglobin antibody. The measurement operation is as follows.

Anti-myoglobin antibody-immobilized beads were charged into a test tube into which 50 μL of a urine sample and 200 μL of a phosphate buffer (100 mM, pH 7.4) were dispensed, and shaken at room temperature for 2 hours (30 rpm). . The reaction mixture was aspirated and the ^{12 5} I-labeled anti-myoglobin antibodies of 200μL was added after washing 3 times with purified water 1 mL, and shaken at room temperature for 2 hours again. The reaction mixture was aspirated and washed 3 times with distilled water 1 mL, bound to the beads ¹²⁵
The radioactivity of the I-labeled antibody was counted. Myoglobin concentration was determined based on measurements using a standard sample instead of a urine sample. Table 1 shows the results.

[0041]

[Table 1]

As shown in Table 1, 6.25, 12.5,
By adding 25, 50, 100, and 200 mM of sodium bicarbonate, the stabilizing effect of urinary myoglobin at 4 ° C. storage was confirmed as compared with the case without sodium bicarbonate. From Table 1, it can be seen that the highest stability was obtained when 400 mM sodium bicarbonate was added, but the measured value was significantly reduced, so 100 mM sodium bicarbonate was used as the optimum concentration.

Example 2 The stabilizing effect of urinary myoglobin was examined in exactly the same manner as in Example 1 except that the cells were frozen and thawed 0, 1, 2, and 3 times instead of storing at 4 ° C. Was. Table 2 shows the results.

[0044]

[Table 2]

As shown in Table 2, when thawed and thawed,
The stabilizing effect of urinary myoglobin was confirmed as in the case of storage at ℃. In this case, as in the first embodiment, 4
It can be seen that the highest stability was obtained when 00 mM sodium bicarbonate was added, but the measured value was significantly reduced.
00 mM sodium bicarbonate was used as the optimal concentration.

Example 3 100 mM sodium bicarbonate was added, and 0,3.
9, 7.8, 15.6, 31.2, 62.5, 125,
Add 250,500 mM sodium azide and add to 4 ° C.
The stabilizing effect of urinary myoglobin was examined in exactly the same manner as in Example 1 except that the cells were stored for 0, 3, and 7 days, respectively. Table 3 shows the results.

[0047]

[Table 3]

As shown in Table 3, it was confirmed that the addition of sodium azide further improved the stabilizing effect of urinary myoglobin as compared with the case where sodium azide was not added. In addition, since a high stabilizing effect was observed by adding sodium azide in the range of 15.6 to 250 mM, an intermediate value of 62.5 mM was determined as the optimum concentration.

Example 4 100 mM sodium bicarbonate was added, and 0,3.
9, 7.8, 15.6, 31.2, 62.5, 125,
Except that 250, 500 mM sodium azide was added
The stabilizing effect of urinary myoglobin was examined in exactly the same manner as in Example 2. Table 4 shows the results.

[0050]

[Table 4]

As shown in Table 4, it was confirmed that the addition of sodium azide further improved the stabilizing effect of urinary myoglobin as compared with the case where sodium azide was not added. In addition, since a high stabilizing effect was observed by adding sodium azide in the range of 15.6 to 125 mM, an intermediate value of 62.5 mM was determined as the optimum concentration.

Example 5 100 mM sodium bicarbonate and 62.5 mM sodium azide were added and 0, 1.25, 2.5,
Stabilizing effect of urinary myoglobin in exactly the same manner as in Example 1, except that 5, 10, 20, 40, 80, and 160 mM of EDTA · 2Na was added and stored at 4 ° C. for 0, 3, and 7 days, respectively. Was examined. Table 5 shows the results.

[0053]

[Table 5]

As shown in Table 5, it was confirmed that the addition of EDTA · 2Na further improved the stabilizing effect of urinary myoglobin as compared with the case where EDTA · 2Na was not added. In addition, since a high stabilizing effect was observed by adding 10 to 80 mM EDTA · 2Na, an intermediate value of 40 mM was determined as the optimum concentration.

Example 6 100 mM sodium bicarbonate and 62.5 mM sodium azide were added and further 0, 1.25, 2.5,
The stabilizing effect of urinary myoglobin was examined in exactly the same manner as in Example 2 except that 5, 10, 20, 40, 80, and 160 mM of EDTA · 2Na was added. Table 6 shows the results.

[0056]

[Table 6]

As shown in Table 6, it was confirmed that the addition of EDTA · 2Na further improved the stabilizing effect of urinary myoglobin as compared with the case where EDTA · 2Na was not added. In addition, since a high stabilizing effect was observed by adding 10 to 80 mM EDTA · 2Na, an intermediate value of 40 mM was determined as the optimum concentration.

Example 7 100 mM sodium bicarbonate, 62.5 mM sodium azide and 40 mM EDTA · 2Na were added,
0.01, 0.025, 0.05, 0.1, 0.2,
Add 0.4, 0.8, 1.6 w / v% Tween 20 and add 4
The stabilizing effect of urinary myoglobin was examined in exactly the same manner as in Example 1 except that it was stored at 0 ° C. for 0, 3, and 7 days, respectively. Table 7 shows the results.

[0059]

[Table 7]

As shown in Table 7, it was confirmed that even if Tween 20 was added, the stabilizing effect of urinary myoglobin was maintained without deterioration.

Example 8 100 mM sodium bicarbonate, 62.5 mM sodium azide and 40 mM EDTA · 2Na were added,
0.01, 0.025, 0.05, 0.1, 0.2,
The stabilizing effect of urinary myoglobin was examined in exactly the same manner as in Example 2, except that Tween 20 of 0.4, 0.8, 1.6 w / v% was added. Table 8 shows the results.

[0062]

[Table 8]

As shown in Table 8, it was confirmed that the addition of Tween 20 further improved the effect of stabilizing urinary myoglobin as compared with the case where Tween 20 was not added. Since a high stabilizing effect was observed by adding 0.01 to 0.2 w / v% of Tween 20, an intermediate value of 0.1 w / v% was determined as the optimum concentration.

COMPARATIVE EXAMPLE 1.5% by weight of sodium azide was added,
1.25, 2.5, 5, 10, 20, 40, 80, 16
The stabilizing effect of urinary myoglobin was examined in the same manner as in Example 2 except that 0 mM EDTA · 3Na was added.
Table 9 shows the results.

[0065]

[Table 9]

As shown in Table 9, in the stabilizer combining sodium azide and EDTA · 3Na, the stabilization shown in Example 6 of the present invention in which sodium hydrogen carbonate, sodium azide and EDTA · 2Na were added. It was confirmed that the stabilizing effect of myoglobin in urine accompanying freezing and thawing was significantly reduced as compared with the preparation.

Claims (28)

[Claims] 1. A method for stabilizing urinary myoglobin, characterized by including a bicarbonate compound in a urine sample. 2. The content of the bicarbonate compound is 6.25 to 2.
The method for stabilizing urinary myoglobin according to claim 1, wherein the concentration is in the range of 00 mM. (3) the bicarbonate compound is sodium bicarbonate;
The method for stabilizing urinary myoglobin according to claim 1 or 2, wherein the method is at least one selected from the group consisting of potassium bicarbonate and ammonium bicarbonate. 4. The method for stabilizing urinary myoglobin according to claim 1, which further comprises an azide. 5. The method for stabilizing urinary myoglobin according to claim 4, wherein the azide content is in the range of 3.9 to 500 mM. 6. The method for stabilizing urinary myoglobin according to claim 4, wherein the azide is at least one selected from the group consisting of sodium azide, potassium azide and ammonium azide. 7. The method for stabilizing urinary myoglobin according to claim 1, further comprising a chelating agent. 8. The method for stabilizing urinary myoglobin according to claim 7, wherein the content of the chelating agent is in the range of 5 to 160 mM. 9. The method for stabilizing urinary myoglobin according to claim 7, wherein the chelating agent is EDTA or a salt thereof. 10. The method according to claim 1, wherein a surfactant is contained.
A method for stabilizing urinary myoglobin according to the above. 11. A surfactant content of 0.01 to 0.2.
The method for stabilizing urinary myoglobin according to claim 10, which is in the range of w / v%. 12. The method for stabilizing urinary myoglobin according to claim 10, wherein the surfactant is a nonionic surfactant. 13. The method for stabilizing urinary myoglobin according to claim 10, wherein the nonionic surfactant is a sorbitan fatty acid ester and / or a polyoxyethylene sorbitan fatty acid ester. 14. The polyoxyethylene sorbitan fatty acid ester is at least one selected from the group consisting of the compounds listed below and derivatives thereof.
14. The method for stabilizing urinary myoglobin according to any one of claims 13 to 13. Polyoxyethylene sorbitan monolaurate Polyoxyethylene sorbitan monopalmitate Polyoxyethylene sorbitan monostearate Polyoxyethylene sorbitan tristearate Polyoxyethylene sorbitan monooleate Polyoxyethylene sorbitan monooleate 15. A stabilizing agent for urinary myoglobin, comprising a bicarbonate compound. 16. The bicarbonate compound is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate.
The stabilizer for urinary myoglobin according to the above. 17. The method according to claim 15, which comprises an azide.
7. The stabilizer for urinary myoglobin according to 6. 18. The stabilizer for urinary myoglobin according to claim 17, wherein the azide is at least one member selected from the group consisting of sodium azide, potassium azide and ammonium azide. 19. A method according to claim 15, which comprises a chelating agent.
8. The stabilizer for urinary myoglobin according to 8. 20. The stabilizer for urinary myoglobin according to claim 19, wherein the chelating agent is EDTA or a salt thereof. 21. The method according to claim 15, further comprising a surfactant.
The stabilizing agent for urinary myoglobin according to 0. 22. The stabilizer for urinary myoglobin according to claim 21, wherein the surfactant is a nonionic surfactant. 23. The stabilizer for urinary myoglobin according to claim 21, wherein the nonionic surfactant is a sorbitan fatty acid ester and / or a polyoxyethylene sorbitan fatty acid ester. 24. The polyoxyethylene sorbitan fatty acid ester is at least one selected from the group consisting of the compounds listed below and derivatives thereof.
24. The stabilizing agent for urinary myoglobin according to any one of claims to 23. Polyoxyethylene sorbitan monolaurate Polyoxyethylene sorbitan monopalmitate Polyoxyethylene sorbitan monostearate Polyoxyethylene sorbitan tristearate Polyoxyethylene sorbitan monooleate Polyoxyethylene sorbitan monooleate 25. A method for immunologically measuring urinary myoglobin, which comprises reacting myoglobin in a urine sample containing a bicarbonate compound with an anti-myoglobin antibody. 26. The method for immunologically measuring urinary myoglobin according to claim 25, comprising an azide. 27. The method for immunologically measuring urinary myoglobin according to claim 25, which comprises a chelating agent. 28. The method for immunologically measuring urinary myoglobin according to claim 25, further comprising a surfactant.