JP2000256399A - Stabilization of myoglobin in urine and stabilization agent therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stabilizing method for myoglobin in urine capable of preventing the lowering of immunologically determined level of myoglobin in urine even after the storage at room temperature as well as the storage at low temperature or the thawing of frozen preparation and to obtain a stabilizing agent to be used therefor. SOLUTION: Myoglobin in urine is stabilized by adding hemoglobin or decomposed hemoglobin and optionally a bicarbonate and/or a chelating agent to a urine specimen. The concentration of hemoglobin and/or decomposed hemoglobin is 0.1-40 mg/mL.

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 immunological measurement of urinary myoglobin even when stored at room temperature in addition to low-temperature storage and freeze-thaw. The present invention relates to a method for stabilizing urinary myoglobin capable of preventing a decrease in the value, and a stabilizer used for the method.

[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 been pointed out that an increase in myoglobin concentration due to muscle tissue damage leads to kidney damage (Clin. Nephrol.,
38, 193-5; 1992).

[0003] Therefore, measuring the myoglobin concentration in urine with high precision is extremely important in diagnosing heart disease and kidney disease. In addition, the pathological diagnosis by measuring urinary myoglobin is different from the measurement of blood myoglobin,
It is non-invasive and will continue to be the recommended diagnostic method.

[0004] However, in the measurement of urinary myoglobin, the stability of myoglobin in a sample poses a problem. In general, it is known that the measurement value of urinary myoglobin significantly decreases when urine is stored at room temperature. Even if urine is frozen immediately after collection, there are samples whose measured values are greatly reduced.

[0005] 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 a urine sample was adjusted to 8.0 to 9.5 with NaOH, and it was confirmed that stable measurement values could be obtained over 12 days even in refrigerated storage.

[0006] However, the pH of the urine sample is adjusted to 8.0 to 8.0.
It became clear that the method of 9.5 was not necessarily effective for all urine samples. That is, although the measured value of myoglobin can be stably maintained 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.

[0007] Furthermore, when a urine sample was frozen, a phenomenon in which the measured value of myoglobin decreased every time the freeze-thaw operation was performed was also confirmed. The phenomenon of decrease in measured values due to freezing and thawing could not be eliminated by the alkali treatment described above.

[0008] Other methods for improving the stability of components in urine samples include the following. 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). When a specific analyte such as methamphetamine is measured, a reaction is performed. An alkaline component is used to suppress the influence of the inhibiting component.

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

However, each of the above publications merely discloses a technique for preventing a reaction-inhibiting phenomenon when measuring a certain kind of analyte in a urine sample, and it stabilizes the storage stability of urinary myoglobin. No gender is suggested.

For the purpose of improving the storage stability of urinary myoglobin, a stabilizer comprising a predetermined amount of sodium azide and EDTA · 3Na has been proposed (JP-A-10-282095).

However, this stabilizing agent can stabilize urinary myoglobin for 3 weeks when stored at 4 ° C., but it has been confirmed that the measured value is significantly reduced by freezing and thawing. Was.

[0013] In order to solve this problem, the applicant of the present invention has proposed a method for freezing and thawing by adding a surfactant in addition to a bicarbonate, an azide (Example: sodium azide) and a chelating agent. Even so, a stabilization method for urinary myoglobin that can prevent a decrease in the urinary myoglobin immunological measurement value and a stabilizing agent used therefor have already been proposed (JP-A-11-60600).

[0014]

However, recently,
Of the azides, sodium azide is designated as a toxicant (decree 405: a decree to revise a part of the toxic and deleterious substances designation order), and generally it is not preferable to use it. .

Accordingly, the present invention has been made in view of such conventional problems, and it is an object of the present invention to stably maintain urinary myoglobin for a long period of time without using the above sodium azide. It is an object of the present invention to provide a method for stabilizing urinary myoglobin and a stabilizer used therefor.

[0016]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that hemoglobin and / or hemoglobin degradation products and, if necessary, bicarbonate and / or a chelating agent in a urine sample. It has been found that by containing, it is possible to stabilize urinary myoglobin for a long period of time not only when stored at low temperature or when frozen and thawed, but also when stored at room temperature, and reached the present invention.

An object of the present invention is to stabilize urinary myoglobin characterized in that a urine sample contains hemoglobin and / or a hemoglobin hydrolyzate and, if necessary, a bicarbonate and / or a chelating agent. This was achieved by the method of stabilization and the stabilizers used therein.

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

The hemoglobin or hemoglobin hydrolyzate used in the present invention is not particularly limited, but is preferably separated and purified from mammalian blood. Specific examples include at least one selected from the group consisting of humans, cows, horses, sheep, goats, rabbits, and pigs. Examples of hemoglobin degradation products include enzyme-treated heme iron.

In the present invention, hemoglobin and / or hemoglobin hydrolyzate is contained in a urine sample in an amount of 0.1 to 40 m.
g / mL, preferably 0.1 mg / mL. If the concentration of hemoglobin or the like is less than 0.1 mg / mL, a sufficient stabilizing effect cannot be obtained, and 40 m
If it exceeds g / mL, no further stabilizing effect can be obtained.

In a urine sample, precipitation may generally occur at a pH around neutrality, and this may cause variations in measured values. It is considered that this precipitate is formed due to factors such as the type and concentration of salts contained in urine, and pH. In the present invention, this precipitation can be prevented by including a bicarbonate and / or a chelating agent in the urine sample, whereby the measured value of urinary myoglobin is kept substantially constant regardless of the sample. Can be maintained.

The bicarbonate used in the present invention can be appropriately selected from known bicarbonates and used.
Specific examples include at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate, and ammonium bicarbonate.

In the present invention, bicarbonate is contained in a urine sample at a concentration of 5.0 to 500 mM, preferably 10 to 100 mM.
M, more preferably 30 mM.
When the concentration of the bicarbonate compound is less than 5.00 mM,
Depending on the urine sample, the stabilizing effect may be insufficient. Conversely, when the amount exceeds 500 mM, precipitation tends to occur, and the measured value is rather lowered.

As described above, a precipitate may be formed due to the type or concentration of salts contained in urine, pH, or the like. This precipitation can also be prevented by including a chelating agent in the urine sample separately from the bicarbonate, and the stabilizing effect of urinary myoglobin can be further improved. However, since this precipitate is not always generated due to salts and pH in urine, a chelating agent may be added as needed.

The chelating agent is contained in the urine sample at 1.00 to 16
0 mM, preferably 5-80 mM, more preferably 40 mM.
It is contained so as to be mM. The concentration of the chelating agent is 1.
When the concentration is less than 00 mM, precipitation occurring in urine cannot be effectively prevented, so that the stabilizing effect becomes insufficient.
If it exceeds 0 mM, the immune response is inhibited, and the measured value is rather reduced.

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, 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.

Although any albumin can be used, albumin derived from animal serum or egg white is particularly preferable. Specific examples include cows, horses, goats, sheep, pigs, rabbits, as well as 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 stabilizer for urinary myoglobin containing hemoglobin or a hemoglobin hydrolyzate that acts as a stabilizer for urinary myoglobin.

The bicarbonate can be suitably 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 a chelating agent. The chelating agent may be appropriately selected from those described above.

The urine sample stabilized with myoglobin 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.

[0034]

According to the present invention, the immunologically measured values of urinary myoglobin can be stably maintained over a long period of time even when stored at room temperature as well as at low temperature or frozen storage.
Therefore, according to the immunological measurement method of the present invention, a heart disease or a kidney disease can be diagnosed with high accuracy.

[0035]

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, 0.2, 0.4, 0.8, 1..
6, 3.2, 6.4, 12.8, 25.6 mg of bovine serum hemoglobin or its decomposed product (heme iron) is added and the mixture is added at 4 ° C.
Were stored for 0, 1, 2, 3, 6, and 9 days, respectively, to examine the effect on the immunological measurement of myoglobin.

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 placed in 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 (30 rpm) at room temperature for 2 hours. . The reaction solution was aspirated, washed three times with 1 mL of purified water, added with 200 μL of ¹²⁵ I-labeled anti-myoglobin antibody, and shaken again at room temperature for 2 hours. After the reaction solution was aspirated and washed three times with 1 mL of purified water, the radioactivity of the ¹²⁵ I-labeled antibody bound to the beads was counted. Myoglobin concentration was determined based on measurements using a standard sample instead of a urine sample. The results are shown in Tables 1 and 2.

[0039]

[Table 1]

[0040]

[Table 2]

As shown in Tables 1 and 2, 0.2,
0.4, 0.8, 1.6, 3.2, 6.4, 12.8,
By adding hemoglobin or a hemoglobin hydrolyzate to 25.6 mg / mL, 4 ° C. was compared with the case where no hemoglobin or hemoglobin hydrolyzate was added.
The stabilizing effect of urinary myoglobin during storage was confirmed. However, it can be seen that even when hemoglobin or a hemoglobin hydrolyzate is added, the measured value varies at each concentration. This is considered to be caused by urine sedimentation due to salts and pH in urine.

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

[0043]

[Table 3]

[0044]

[Table 4]

As shown in Tables 3 and 4, the stabilizing effect of urinary myoglobin was confirmed also in the case of freezing and thawing as in the case of storage at 4 ° C. However, an increase in the measured value due to freeze-thaw caused by urine sedimentation is observed.

Example 3 To 1 mL of a urine sample, 0, 1, 2, 4 mg of hemoglobin or a hemoglobin hydrolyzate (heme iron) was added, and the mixture was heated to 37 ° 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 and 1 day, respectively, and stored at 4 ° C. and 25 ° C. for 4 days. Table 3 shows the results.

[0047]

[Table 5]

As shown in Table 5, the stabilization effect of urinary myoglobin was confirmed not only at 4 ° C. but also at room temperature as compared with the case where hemoglobin or hemoglobin hydrolyzate was not added.

Example 4 1 mg of hemoglobin, 16.8 per 2 mL of urine specimen
mg sodium bicarbonate and 29.78 mg ED
Providing a urine collection tube was added TA2Na · 2H ₂ O, and collected urine 6 specimens, 4 ° C. to 0, 1, 2, 3, 4, except that was stored for 7 days, in the same manner as in Example 1 The stabilizing effect of urinary myoglobin was examined. As a control, those without these stabilizers were similarly examined for the stabilizing effect.
The results are shown in Tables 6 and 7, respectively.

[0050]

[Table 6]

[0051]

[Table 7]

As shown in Table 6, it was found that the sample to which the stabilizer of the present invention was added exhibited a high stabilizing effect regardless of the storage days. On the other hand, it can be seen that the measured value of the sample to which the stabilizer was not added was remarkably reduced with the lapse of the storage days (see Table 7). In addition to hemoglobin or hemoglobin degradation product, by adding EDTA2Na · 2H 2 _O of sodium bicarbonate and the measured value without vary, it can be seen that a substantially constant measured value was obtained (Table 1 and See Table 2). This means
As described above, it is considered that urine sedimentation caused by salts and pH in urine could be prevented.

Example 5 A urinary tract was prepared in the same manner as in Example 4, and six samples of urine were collected and freeze-thawed 0, 1, 2, 3, 4, and 5 times.
The stabilizing effect of urinary myoglobin was examined in exactly the same manner as in Example 4. The results are shown in Tables 8 and 9, respectively.

[0054]

[Table 8]

[0055]

[Table 9]

As shown in Table 8, it can be seen that the sample to which the stabilizer of the present invention was added obtained a substantially constant measured value regardless of the number of freeze-thaw cycles. On the other hand, in the sample to which the stabilizer was not added, the measured value was found to be significantly reduced every time the freeze-thaw was repeated (Table 9). In addition to hemoglobin or hemoglobin degradation product, by adding EDTA2Na · 2H 2 _O of sodium hydrogen carbonate and, without the measured value is increased, it can be seen that a substantially constant measured value was obtained (Table 3 And Table 4). This is also considered to be because urinary sedimentation caused by salts and pH in urine was prevented as described above.

Claims (11)

[Claims] 1. A method for stabilizing urinary myoglobin, characterized in that hemoglobin and / or hemoglobin hydrolyzate is contained in a urine sample. 2. The method for stabilizing urinary myoglobin according to claim 1, wherein the concentration of hemoglobin and / or hemoglobin hydrolyzate is in the range of 0.1 to 40 mg / ml. 3. The method for stabilizing urinary myoglobin according to claim 1, further comprising a bicarbonate and / or a chelating agent. 4. The concentration of bicarbonate is from 5.0 to 500 mM.
The method for stabilizing urinary myoglobin according to claim 3, which is in the range of: 5. The method for stabilizing urinary myoglobin according to claim 3, wherein the bicarbonate is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate. 6. The method for stabilizing urinary myoglobin according to claim 3, wherein the concentration of the chelating agent is in the range of 1 to 160 mM. 7. The method for stabilizing urinary myoglobin according to claim 3, wherein the chelating agent is EDTA or a salt thereof. 8. A stabilizing agent for urinary myoglobin, comprising hemoglobin and / or a hemoglobin hydrolyzate. 9. The stabilizer for urinary myoglobin according to claim 8, further comprising a bicarbonate and / or a chelating agent. 10. The stabilizer for urinary myoglobin according to claim 9, wherein the bicarbonate is at least one selected from the group consisting of sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate. 11. The stabilizer for urinary myoglobin according to claim 9, wherein the chelating agent is EDTA or a salt thereof.