JPH08122329A - Composition for quality control and/or accuracy control of lithmus paper for detection of white blood corpuscle in urine as well as quality control method and/or accuracy control method - Google Patents

Abstract

PURPOSE: To execute a control operation easily, quickly and precisely by a method wherein a composition, for control, which is used to execute the quality/ accuracy control operation of lithmus paper is formed of a hydrolase. CONSTITUTION: An esterase or a protease can be used as a hydrolase, a cholinesterase is preferable as the esterase, but they can be used singly or by mixing two or more kinds. The hydrolases are dissolved in purified water or in a proper slow-acting solution so as to decide their activity value. For example, in the case of a butyryrocholinesterase, a butyrylcholin is used as a substrate, an enzyme amount which hydrolyzes 1μmol of the butyrylcholin in one minute at 37 deg.C in a 0.2M phosphoric acid buffer (pH: 8.0) is used as one unit, and an activity value at 500 units/ml or lower is preferable as the hydrolases having a white-blood-corpuscle concentration corresponding to several to several hundred pieces/μl. Since the compositions for a sample always display a constant activity value, they are effective as compositions for control in the quality/accuracy control operation of lithmus paper.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a control composition used for quality control and / or quality control in a test strip for detecting leukocytes in urine, and quality control and / or accuracy using the composition. It concerns management methods.

[0002]

2. Description of the Related Art Currently, as a test strip for detecting leukocytes in urine, Uri Fret II-9 manufactured by Kyoto Daiichi Kagaku Co., Ltd.
C, BM test 10 by Boehringer Mannheim, N-Multistics SG-L by Miles Sankyo,
Lapingnost Total Screen L from Behringberge is commercially available (all of which are registered trademarks), but all of these test papers detect leukocytes in urine by measuring the esterase activity of leukocytes. . The basic components contained in the test strip are a substrate for measuring esterase activity and a diazonium salt as a color-developing agent for detecting a hydrolyzate of the substrate, although the type varies slightly depending on the manufacturer. The amount of white blood cells can be determined by measuring the degree of color development caused by the coupling between the hydrolyzate of the substrate and the color former.

At the time of manufacturing these test papers, quality control of intermediate products and final products is required. In addition, performance evaluation such as accuracy and reproducibility, lot management, degree of sensitivity deterioration, and accuracy management such as fluctuations related to inspection procedures are also required.

[0004] At present, a method using urine containing white blood cells obtained from an actual patient (hereinafter sometimes referred to as white blood cell-containing urine) as a control composition for the management of these, whole blood A method using urine containing separated and purified white blood cells and a method using commercially available control urine are known.

[0005]

The management methods as described above, which are currently in use, have the following drawbacks.

In the method of using urine containing white blood cells obtained from an actual patient as a control composition, the component of urine itself (that is, the portion excluding the white blood cells) interferes with the measurement, and thus the esterase activity of the white blood cells is accurately measured. No method has been established, and it is difficult to prepare a solution that constantly exhibits a constant esterase activity value. Also, it is difficult to maintain a constant value of esterase activity for a long period of time due to the deterioration of white blood cells.

In the method using leukocytes separated and purified from whole blood, in addition to the same problem of activity retention as the above-mentioned leukocyte-containing urine, there is a problem that much labor and time are required for obtaining whole blood and separating and purifying it from whole blood. There is. There is also a risk of infection due to the use of whole blood.

In the method using commercially available control urine,
The process of color development is not the same as the detection principle of test strips. That is, the esterase activity-like substance in the control urine does not hydrolyze the substrate, and the hydrolyzate and the diazonium salt do not undergo a coupling reaction to develop color. Actually, the pseudo substance and the diazonium salt, which is the color former, are only reacted and colored, and it cannot be said that they are truly quality control and / or precision control.

The present invention solves these problems of the prior art, and easily, swiftly and accurately carries out quality control and / or quality control for a test strip for detecting leukocytes in urine. The present invention provides a control composition and a method for quality control and / or quality control using the control composition.

[0010]

The first invention for solving the problems has been described above as a sample composition for quality control or / and quality control of test strips for detecting leukocytes in urine. Rather than using leukocytes with the same problems or commercially available control urine, it is useful as a control substance whose activity can be easily valued using the same measurement principle as when using leukocytes, and is easily available. A safe and control composition comprising a hydrolase.

The second invention is a quality control and / or quality control method using the control composition of the first invention.

As the hydrolase of the present invention, esterase and protease can be used. The esterase is preferably cholinesterase and the like, and the protease is preferably chymotrypsin, trypsin, elastase, subtilisin, thrombin, plasmin, cathepsin, papain, kallikrein and the like,
It is not limited to these hydrolases. These hydrolases may be used alone or in combination of two or more. Generally, these hydrolases are easily available as reagents.

These hydrolases are dissolved in purified water or an appropriate buffer solution, and the activity value of the enzyme solution is determined by a generally known enzyme activity value measuring method. For example, butylcholinesterase (manufactured by Sigma, product number C-5386,
In the case of human serum-derived EC3.1.1.8), butyrylcholine is used as a substrate, and 0.2 M phosphate buffer (pH 8.
In 0), the amount of the enzyme that hydrolyzes 1 μmol of butyrylcholine in 1 minute at 37 ° C. is 1 unit (hereinafter referred to as unit). Elastase (Product number E012 manufactured by Sigma)
7, porcine pancreas origin, EC3.4.21.36),
Using elastin as a substrate, 0.1M Tris-HCl buffer (p
In H8.8), the amount of enzyme that solubilizes 1 mg of substrate in 37 minutes at 37 ° C. is 1 unit. Subtilisin (Boehringer Mannheim, EC 3.4.21.14)
In the case of N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (N-su
ccinyl-Ala-Ala-Pro-Phe-p-
Nitroanilide) as a substrate, and 0.1 μmol of p / min is added in 0.1 M Tris-HCl buffer (pH 7.8) for 1 minute.
-The amount of enzyme that releases nitroaniline (p-nitroaniline) is 1 unit.

A test strip for detecting leukocytes in urine usually needs to detect several to several hundreds / μl of leukocytes in urine. Of course, higher concentrations of specimens are also present, so the test strips need to be inspected to ensure that they do not cause the prozone phenomenon. Therefore, the solution of the control composition is required to have a white blood cell concentration of several to several hundreds / μl, and a very wide range corresponding to a high concentration.

Therefore, the activity value of the control composition cannot be unconditionally limited, but, for example, when the leukocyte concentration is equivalent to several to several hundreds / μl, in the case of cholinesterase, it is 500 units / ml or less, that of chymotrypsin. In the case of (manufactured by Sigma, product number C7762, calculated from the activity notation specified by the manufacturer) 200 units / ml or less, in the case of trypsin (manufactured by Sigma product number T864
2, calculated from the activity notation specified by the manufacturer) 50000un
its / ml or less, 60 units for elastase
s / ml or less, 150 units for subtilisin
/ Ml or less, cathepsin B (manufactured by Sigma, serial number C62
86, calculated from the activity notation specified by the manufacturer) 3500un
It is preferably below its / ml. When an activity value corresponding to a high white blood cell concentration of several hundreds / μl or more is required, an enzyme solution having the above-mentioned activity value range or higher activity value range may be prepared. Needless to say, the unit number is higher than 0.

A stabilizer may be added for stabilizing the enzyme solution. As stabilizers, commonly used substances include buffers, sugars, cation compounds,
There are proteins, surfactants, activators, reversible inhibitors, etc.

Examples of the buffering agent as a stabilizer include citric acid-sodium hydroxide, potassium hydrogen phthalate-hydrochloric acid, glycine-hydrochloric acid, acetic acid-sodium acetate, succinic acid-sodium hydroxide, disodium hydrogen phosphate- Sodium dihydrogen phosphate, dipotassium hydrogen phosphate-sodium hydroxide, Tris-hydrochloric acid, Bicine-sodium hydroxide, boric acid-sodium hydroxide, sodium borate-sodium hydroxide, disodium carbonate-hydrogen carbonate Sodium, 5,5-diethylbarbituric acid sodium-hydrochloric acid, 2-amino-2-methylpropane-1,3-diol-hydrochloric acid and the like can be mentioned. Examples of saccharides include trehalose, sucrose, maltose, lactose, inositol and the like. Examples of the cation compound include calcium ion and magnesium ion, and examples of the protein include albumin. Examples of the surfactant include nonionic surfactants such as octyl-D-glucoside, polyethylene glycol lauryl ether and saccharose. Examples include monodecanoate. Examples of the activator include glutathione, cysteine, 2-mercaptoethanol, dithiothreitol and the like, and reversible inhibitors such as phenol, indole and hydrocinnamic acid can also be used. The stabilizer is not limited to these compounds, and the stabilizer may be used alone or as a mixture of two or more kinds.

When a stabilizer is added, the concentration with respect to the enzyme solution is preferably 0.01 to 1 mol / l for a buffer and 0.1 to 50% for a saccharide, preferably 1 ~ 20%, 0.0 for cation compounds
1-1 mol / l, 0.1-5% for proteins, 0.01-1% for surfactants, and 0.1% for activators.
001 to 1 mol / l, 0.001 for reversible inhibitors
It is preferable to add ~ 1 mol / l.

Except for substances vulnerable to low temperature, generally, the solution can be used for a long period of time if it is refrigerated or stored frozen. However, when storing for a longer period of time or considering convenience,
A vacuum freeze-dried product is effective. These enzyme solution samples according to the present invention can also be processed into vacuum freeze-dried products in order to improve storage stability. Vacuum freeze-drying may be performed under the conditions generally performed.

For example, a vacuum freeze dryer (DR-03H type manufactured by Nippon Vacuum Technology Co., Ltd.) is used. Into a 5 ml vial add 3 ml of the enzyme solution priced as above and freeze completely at -20 ° C. The pressure is reduced to 1 mmHg or less by a vacuum pump, and the cooling is stopped when the degree of pressure reduction becomes constant. The pressure reduction is continued, and after the temperature of the sample rises to room temperature, the pressure is further reduced for half a day and the drying is completed. Leak with nitrogen or dry air and seal with a rubber stopper to make a vacuum freeze-dried product. In this case, the enzyme solution to be frozen preferably contains the above-mentioned stabilizer. The concentration is also as described above.

These freeze-dried products are used as a control composition solution by adding 3 ml of purified water or a buffer at the time of use and mixing by inversion. As a solution for dissolving the dried product, in some cases, necessary additives (for example, a stabilizer for the enzyme solution, a coloring substance in other items, etc.) may be added to the side of the solution to obtain a specified solution. . This will be described later.

Considering the stability during storage in a solution state,
The optimum pH of the enzyme should be considered. In the case of protease, the pH of the buffer solution for dissolving the protease should be shifted to the acidic or alkaline side within a range of about 0.5 to 3.0 than the optimum pH of the activity of the protease in order to avoid self-digestion. However, long-term storage is possible even after returning to a solution state.

By the way, a test strip for detecting leukocytes in urine is usually commercially available as a multi-item test strip which can be measured simultaneously with other components in urine. For example, in the case of Uri Fret II-9C, occult blood including white blood cells, urobilinogen, bilirubin, protein, glucose, ketone, nitrite,
In order to be able to measure 9 items of pH at the same time, the test paper part in which each measuring reagent is impregnated on the base plastic sheet is separated and attached as one stick-shaped piece. Therefore, it is desirable that all of these measurement items can be quality controlled and / or quality controlled at the same time.

That is, a substance capable of simultaneously measuring these items may be added to a control sample for quality control and / or quality control of a test strip for detecting leukocytes in urine. For example, bovine blood hemoglobin for occult blood, bovine serum albumin for proteins, glucose for glucose, acetone for ketones, urobilinogen for urobilinogen and its pseudo-substance naphthresorcin, naftrezorcin for bilirubin and zitaurobilirubin for bilirubin,
And sodium nitrite for nitrous acid, uric acid and sodium chloride for specific gravity. Needless to say, these concentrations are predetermined.

[0025]

Since the sample composition of the present invention always exhibits a constant activity value, it is very useful as a control composition for quality control and / or quality control of test strips for detecting leukocytes in urine. Became useful to. Because the enzyme is used instead of using real white blood cells or commercially available control urine, it is safe without blood infection, the material is easily available, and the measurement principle is the same as when white blood cells are used. The value has also become more reliable. Further, even when processed into a frozen product or a vacuum freeze-dried product, the activity as a control substance did not decrease, and the storability and convenience were also improved.

Example 1 Preparation of Control Solution According to the Present Invention Porcine pancreatic elastase (manufactured by Sigma, product number E0127,
Lot 121H8090) was dissolved in Tris buffer (0.1 M Tris-HCl buffer, pH 8.8, containing 0.01 M calcium chloride) to give 50 mg / l. Manufacturer-specified activity assay (using substrate elastin, pH 8.8, 3
The activity was measured by using 1 unit as the amount of the enzyme that solubilizes 1 mg of elastin for 20 minutes at 7 ° C. or lower, and the result was 3.5 units / ml. Minus 3 of this solution
It was stored frozen at 0 ° C.

Preparation of test paper (white blood cells) Kyoto Daiichi Kagaku Yuri Fret II-9C (special machine SA-
4230), BM of Boehringer Mannheim
Test 10 (visual, room temperature, 1 minute), Miles Sankyo's N-Multistics SG-L (visual, room temperature, 1 minute), Beringberke's La Pygnost Total Screen L (visual, room temperature, 1 minute) Using.

Preparation of Urine Samples Containing Leukocytes In urine sedimentation, 5 samples in which 10 to 100 leukocytes were detected per strong field were pooled to obtain leukocyte-containing urine.

Color development test The color development of the above test papers was compared using a thawed solution of the control solution and a urine sample containing white blood cells as samples. However, in the case of Uri Fret II-9C, the judgment was made by visual observation for 1 minute (37 ° C.). The results are shown in Table 1. It can be seen that the white blood cell solution and the control solution show the same color, and the control solution shows the same reactivity as the white blood cells on the test paper.

Simultaneous reproducibility test The above control solution was melted and repeatedly measured 10 times with the above test paper. The results are shown in Table 2. Good simultaneous reproducibility was obtained. It can be seen that this solution is a good control solution.

When the day difference reproducibility is used, the above control solution is thawed, and Yuri Fret II is used.
The reflectance was measured by repeating -9C (dedicated measuring instrument SA-4230) three times at approximately the same time for 5 days.

The results are shown in Table 3. It can be seen that good day reproducibility was obtained, and that this solution has good performance as a control solution for quality control and / or quality control of test paper for detecting white blood cells.

Example 2 Preparation of Control Solution (Sample Composition) According to the Present Invention Subtilisin (Boehringer Mannheim, Lot No. 13399123-32, 13541220-3)
3) was added to Tris buffer (0.1 M Tris-HCl buffer, p
H7.8, containing 0.01M calcium chloride)
It was set to 20 mg / l. N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (N-succinyl-Ala-Ala) as a substrate
-Pro-Phe-p-nitroanilide) at a pH of 7.8 and 30 ° C. to release p-nitroaniline (p-nitroaniline) at 41%.
It was measured as the absorbance at 0 nm. 1 μmo per minute
le's p-nitroaniline (p-nitroanili)
The amount of the enzyme that releases ne) was set to 1 unit.

As a result, the enzyme of lot 13399123-32 was 1.14 unit / ml, and the lot 13399123-32.
The enzyme of -34 became 0.94 unit / ml. And 1.5 units / ml of these subtilisin lots
Was prepared so that

Preparation of test paper (white blood cells) Yuri Fret II-9C (special machine SA- of Kyoto Daiichi Kagaku)
4230) was used.

[Effect test of difference in lots of subtilisin on test paper sensitivity] Two lots of subtilisin were used, and 1.5 units were used for each.
/ Ml control solution was prepared and
The measurement was repeated 0 times. The results are summarized in Table 4. A control solution was prepared using two lots with different activity values per unit weight of subtilisin. By matching the activity values, it is possible to prepare a control solution that always shows the same sensitivity with test paper and detect leukocytes. It can be seen that the sample has good performance as a sample for quality control and / or quality control of the test paper.

Example 3 Preparation of Vacuum Freeze-Dried Product According to the Present Invention Subtilisin (Boehringer Mannheim Co., Lot No. 13729722-34) was added to Tris buffer at 1.5 unit / ml based on Example 2. Dissolved (solution A). Trehalose was added to and dissolved in the subtilisin solution at a volume ratio of 2% (solution B).
Solution A or Solution B 7 ml in a 10 ml vial
Was added, and the samples were placed on a sample shelf cooled to -20 ° C and frozen.

After confirming that the temperature of the sample became -20 ° C, a vacuum freeze dryer (DR-03H manufactured by Nippon Vacuum Technology Co., Ltd.) was used.
Type) vacuum pump was activated. The pressure was reduced to 1 mmg or less, cooling was stopped when the degree of pressure reduction became constant, and the pressure reduction was continued overnight. After the temperature of the sample was raised to room temperature, the pressure was further reduced for half a day, and the drying was completed. It was leaked with nitrogen, sealed with a rubber stopper and left at storage temperatures of 25 ° C and 40 ° C.
The vacuum freeze-dried product obtained from solution A is called freeze-dried A, and the solution B is freeze-dried B. As a reference, minus 30 of solution A
A product stored at ℃ was used.

[Activity measuring method] At the time of use, 7 ml of purified water was added to freeze-dried A and B and mixed by inverting, and the activity was measured in the same manner as in Example 2. The reference solution A stored at −30 ° C. also melted at the time of use, and the activity was measured in the same manner.

Preparation of test paper (white blood cells) Kyoto Daiichi Kagaku Yuri Fret II-9C (special machine SA-
4230) was used.

Stability test results for frozen and freeze-dried products Table 5. The test results are summarized in. It can be seen that the frozen product stored at -30 ° C retains the activity stably for 2 months and is sufficiently effective as a sample for quality control and / or quality control of test paper for detecting leukocytes. Further, the processed product into a vacuum freeze-dried product was stable, and freeze-dried B to which trehalose was added as a stabilizer showed good performance of being stable for 2 months even under a severe condition of 40 ° C.

[0042]

[Table 1]

[0043]

[Table 2]

[0044]

[Table 3]

[0045]

[Table 4]

[0046]

[Table 5]

[0047]

[Table 5]

Claims (14)

[Claims] 1. A control composition for performing quality control and / or quality control on a test strip for detecting leukocytes in urine, wherein the composition comprises a hydrolase. A control composition for quality control and / or quality control. 2. The composition according to claim 1, wherein esterase is used as a hydrolase. 3. The composition according to claim 1, wherein a protease is used as a hydrolase. 4. The composition according to claim 3, wherein elastase is used as the protease. 5. The composition according to claim 3, wherein subtilisin is used as the protease. 6. The composition according to claim 3, wherein chymotrypsin is used as the protease. 7. A quality control method comprising using a control composition comprising a hydrolase as a control composition used for quality control and / or quality control of a test strip for detecting white blood cells in urine. And / or quality control methods. 8. The method according to claim 7, wherein the hydrolase comprises esterase. 9. The method according to claim 7, wherein the hydrolase comprises a protease. 10. The method according to claim 9, wherein the protease comprises elastase. 11. The method according to claim 9, wherein the protease comprises subtilisin. 12. The method according to claim 9, wherein the protease comprises chymotrypsin. 13. The composition according to any one of claims 1 to 6, characterized in that the composition is a vacuum freeze-dried product. 14. The composition according to claim 13, wherein the vacuum freeze-dried product contains a stabilizer.