JP2002243728A - Method of stabilizing protein and peptide in urine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of stabilizing protein and peptide in urine, and a composition therefor. SOLUTION: The protein and the peptide in the urine is stabilized and an accurate measured value is provided, by adding an ionic surfactant, a cabonate and/or a hydrogencarconate of this method into an urine specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stabilizing method for stably maintaining urinary proteins and urinary peptides to be measured for a long time, a composition used therefor, and a stabilized urinary protein or urinary peptide. Related to the measurement method.

[0002]

2. Description of the Related Art Diagnosis of pathological conditions by quantifying urinary protein and urinary peptide (hereinafter collectively referred to as urine protein) is based on the fact that there is no burden on patients due to blood sampling and the risk of infection with hepatitis and AIDS is low. This is a diagnostic method that will be increasingly developed in the future. The urine protein used for diagnosis, a marker of insulin secretion C- peptide basic fetal protein is a cancer marker, renal after hemorrhagic marker alpha ₂ macroglobulin, drop and tubular damage of renal blood flow reflecting the beta ₂ microglobulin, further parathyroid hormone, fibronectin and the like.

However, a problem in the measurement of these urinary proteins is the stability of the proteins in storing urine specimens. Urine contains much less protein than serum, and analytes have a weak protective effect. It is also known that the pH tends to fluctuate, and the stability of urinary protein is significantly reduced in acidic urine. Freezing urine immediately after collection stabilizes the protein in urine, but it requires time for thawing at the time of measurement, and the work is too complicated and difficult to perform in 24-hour urine where urine is collected and accumulated at regular intervals. There's a problem.

[0004] Therefore, the addition of bovine albumin and the addition of bicarbonate to urine (JP-A-11-60600, JP-A-2000-2000)
-221190), addition of reducing oxyacid salt and / or isothiazolone-based compound (JP-A-2000-352565)
Attempts have been made to add sodium azide as a preservative. However, these methods have not been sufficient for stabilizing some urinary proteins.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for stabilizing urinary protein and a composition therefor.

The present invention has been made in view of the above problems, and provides a stabilization technique which does not require a special reagent and is particularly suitable for a method for immunologically measuring urine protein. Aim.

[0007]

This problem is solved by adding an ionic surfactant to a urine sample. As a result of intensive studies, the present inventors have found that the protein in urine is stabilized by adding the above substances to urine. Furthermore, they have found that carbonates and / or bicarbonates have a synergistic stabilizing effect with ionic surfactants, and have completed the present invention.

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

The ionic surfactant used in the present invention comprises:
It can be appropriately selected from known ones and used. As a specific example, sodium dodecyl sulfate (S
DS), dodecyltrimethylammonium chloride,
Examples include anionic surfactants such as triethanolamine lauryl sulfate and sodium lauroyl sarcosinate, and cationic surfactants such as dodecyltrimethylammonium bromide and cetyltrimethylammonium bromide (CTAB). These are also surfactants frequently used in the DNA extraction step.

By adding one or more of these compounds to a urine sample, an effect of stabilizing urinary proteins can be obtained. The addition amount is preferably in the range of 0.005 to 2% by weight, and more preferably in the range of 0.01 to 0.2% by weight. If it is less than 0.005%, the stabilizing effect cannot be obtained, and if it is 2% or more, there is a problem in solubility. Further, a non-ionic surfactant such as Triton X-100 or Tween 20 may be added to these at a concentration of 0.1% by weight or less.

The mechanism of the stabilizing effect of urinary proteins by ionic surfactants is unknown, but does not maintain urine pH constant. This is because in the case of acidic urine, a sample having insufficient stabilizing effect is slightly observed. For the purpose of maintaining the pH of urine at a weak alkali, it is preferable to add a carbonate or a bicarbonate. As described above, carbonates and bicarbonates have a urinary protein stabilizing effect that is more than simply maintaining pH. By using a carbonate or bicarbonate in combination with an ionic surfactant, the protein stabilizing effect in urine is synergistically improved.

The carbonate is selected from sodium carbonate, potassium carbonate, sodium potassium carbonate and ammonium carbonate, and the hydrogen carbonate is selected from sodium hydrogen carbonate, potassium hydrogen carbonate and ammonium hydrogen carbonate. Good. The addition concentration is 10 to 500
It is contained in the range of mM, preferably 25 to 250 mM.

[0013] Preservatives may be further added to inhibit the growth of bacteria in urine. As the preservative used in the present invention, an azide having excellent solubility is preferable. Specific examples of azides include sodium azide, potassium azide and ammonium azide. The addition concentration is
Less than 0.2% is preferred.

By adding the above stabilizer, urinary protein is converted into C-peptide, a marker of insulin secretion amount,
Basic fetal protein cancer markers, alpha ₂ macroglobulin postrenal bleeding markers, beta ₂ microglobulin to reflect the reduction and tubular damage of renal blood flow, over further fibronectin, a variety parathyroid hormone (PTH) It was suggested that most of the protein in urine was effective.

The method of using the stabilizer of the present invention is as follows. A sample tube is weighed in advance and a specified amount of urine is injected. 2. The concentrate prepared in advance is dropped onto the test tube sample.
And so on. In the case of using a concentrated solution, volume correction is required, whereas in the case of a powdered reagent, the volume increase is within a negligible range, and volume correction is not particularly required. For example, if powdered reagents in which SDS and sodium carbonate are respectively weighed and mixed homogeneously are subdivided into test tubes, they can be used immediately when needed. In the case of an animal urine sample, urine may be added to a container in which 2 L of the stabilizer is subdivided. Although 24-hour urine fluctuates in the range of 1 to 3 L, since the stabilizer of the present invention has a wide effective concentration range, the stability of urinary protein is not affected by an increase or decrease in the amount of animal urine.

The urine sample in which urine protein is stabilized according to the present invention can be directly used as a sample for measuring urinary protein based on a known immunological measurement method. The measurement method is not particularly limited,
Immunological agglutination, competition, RIA, IRMA, EI
A, it can be applied to all existing immunological measurement methods such as ELISA. The urine sample has no effect on urinary creatinine measurements by the Jaffe method or the enzymatic method.

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

[0018]

Industrial Applicability The present invention provides urine protein and urine peptide,
In particular, immunological measurement values of various markers of high diagnostic value can be stably maintained over a long period of time. Therefore, if the sample according to the present invention is used for immunological measurement, various diseases and the like can be diagnosed with high accuracy.

[0019]

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, Stabilizing effect of SDS on C-peptide 0, 0.005, 0.01, 0.
05, 0.1, 0.5, 1.0, 2.0%
DS (Daiichi Pure Chemicals) was added, and the mixture was allowed to stand at 25 ° C. for 1 day to examine the effect of urinary C-peptide on immunological measurement.

For the immunological measurement, a commercially available assay kit for IRMA of C-peptide “Ab beads C-peptide '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-C-peptide antibody. The measurement operation is as follows.

A diluent (100 mM phosphate buffer, p
H6.0), 50 μL of urine sample diluted 21-fold with 125 I
The beads immobilized with the anti-C-peptide antibody are introduced into a test tube into which 200 μL of the labeled antibody has been dispensed, and shaken at room temperature for 3 hours (3.
0 rpm). The reaction solution was aspirated, washed three times with 2 mL of purified water, and the radioactivity of each test tube was measured using a well-type scintillation counter. The C-peptide concentration was calculated based on the measured value using a standard sample instead of a urine sample. Table 1 shows relative values when the measured value of the cryopreserved sample as a control was set to 100.

[0023]

[Table 1] Relative measured value of C-peptide (unit relative%) ── SDS (%) 0 0.005 0.01 0.05 0.1 0.5 1.0 2.0 ──────────────────────────────────── Sample 1 43 65 84 98 95 91 94 92 Sample 2 19 87 101 106 97 90 103 100 Sample 3 92 98 101 101 103 97 94 95 Sample 4 39 104 113 114 108 110 101 103 Sample 5 90 96 101 100 97 90 100 98 ────────────────────────────────────

As shown in Table 1, SDS was 0.005%
By containing the above, urine C in storage at 25 ° C.
-A stabilizing effect of the peptide was confirmed. If it is 0.01% or more, almost the same stabilizing effect can be obtained, but considering the ease of dissolution, 0.01 to 0.2% was determined as the optimum concentration.

Embodiment 2 FIG. Stabilizing Effect of Combination of SDS and Sodium Carbonate The stability of urinary C-peptide was tested under the same conditions as in Example 1 except that sodium carbonate was added to 0.1% SDS to a concentration of 0,50,100 mM. . Table 2 shows the results.

[0026]

Table 2 Relative measured value of C-peptide (unit: relative%) ─ Na ₂ CO ₃ (mM) 0 10 25 50 100 250 500 ────────────────────────────────── Sample 6 84 91 99 102 101 103 102 Sample 7 76 85 101 100 102 99 100 Sample 8 101 103 106 101 100 104 105 Sample 9 103 100 98 101 104 102 97 Sample 10 92 98 103 106 105 105 104 ────── ────────────────────────────

[0027] It was confirmed that the addition of sodium carbonate stabilizes a specimen whose stability was insufficient with SDS alone.

Embodiment 3 FIG. Urinary beta ₂ - to stabilize the test urine samples microglobulin (β ₂ -M), 0.01~1.0%
After addition of CTAB and 50 mM sodium bicarbonate, the mixture was allowed to stand at 25 ° C. for 1 day, and then the β ₂ -M value was determined by an ordinary method using LA reagent β ₂ -M (Eiken Chemical Co., latex agglutination method). Table 3 shows the results.

[0029]

[Table 3] β ₂ -M measured value ──────────────────────────────── Storage condition Freezing 25 ℃ ・ 1 day Left ──────────────────────────────── CTAB concentration 0% 0% 0.01% 0.1% 1.0% ───── ─────────────────────────── NaHCO ₃ concentration 0 mM 0 mM 50 mM 50 mM 50 mM ─────────────── ───────────────── Sample 11 (μg / L) 2088 1904 2133 1989 2260 (%) 100% 91% 102% 95% 108% ──────検 体 Sample 12 (μg / L) 2005 1680 2168 2055 2136 (ratio /%) 100% 84% 108% 102 % 107% 検 体 Sample 13 (μg / L) 2310 1250 2760 2273 2360 %) 100% 54% 120% 98% 102% ─ ──────────────────────────────

A stabilizing effect on urinary β ₂ -M was also confirmed with the combination of the cationic surfactant CTAB and sodium bicarbonate. These results indicate that the addition of an ionic surfactant and / or a carbonate and / or bicarbonate to a urine sample stabilizes urine protein or urine peptide.

Claims (12)

[Claims] 1. A method for stabilizing urinary protein and urine peptide, comprising incorporating an ionic surfactant into a urine sample. 2. An ionic surfactant comprising sodium dodecyl sulfate, dodecyltrimethylammonium chloride,
The method for stabilizing urinary protein and urine peptide according to claim 1, wherein at least one selected from the group consisting of triethanolamine lauryl sulfate, sodium lauroyl sarcosinate, dodecyltrimethylammonium bromide and cetyltrimethylammonium bromide is selected. 3. The method according to claim 1, wherein the concentration of the ionic surfactant is 0.005.
The method for stabilizing urinary protein and urine peptide according to claim 1, wherein the amount is in the range of 2 to 2% by weight. 4. The method for stabilizing urinary protein and urine peptide according to claim 1, which comprises a carbonate and / or a bicarbonate. 5. The carbonate or bicarbonate is at least one selected from the group consisting of sodium carbonate, potassium carbonate, sodium / potassium carbonate, sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate. A method for stabilizing urinary protein and urine peptide according to the above. 6. The content of carbonate or bicarbonate is 10 to 5
The method for stabilizing urinary protein and urine peptide according to claim 4, wherein the amount is in the range of 00 mM. 7. The method according to claim 7, wherein the urine protein and the urine peptide are C-pepti.
, Basic fetal protein, α _Two-Macroglobulin, β_Two-Ma
Iglobulin, parathyroid hormone and fibrone
7. The method according to claim 1, which is selected from the group consisting of Kuching.
A method for stabilizing urinary protein and urine peptide. 8. The method according to claim 1, wherein the concentration of sodium dodecyl sulfate is 0.0
The method for stabilizing urinary C-peptide according to claim 1, wherein the amount is 1 to 1.0% by weight. 9. A stabilizer for urinary proteins and urine peptides, which comprises an ionic surfactant. 10. The urinary protein and urinary peptide stabilizer according to claim 9, which comprises an ionic surfactant and a carbonate and / or bicarbonate. 11. A method for immunologically measuring urine protein or urine peptide, which comprises reacting a protein or peptide in a urine sample containing an ionic surfactant with an anti-protein antibody or anti-peptide antibody. 12. The method for immunologically measuring urinary protein and urine peptide according to claim 11, comprising an ionic surfactant and a carbonate and / or bicarbonate.