JP2739484B2 - Urea nitrogen measurement reagent for automatic analyzer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a measurement of urea nitrogen for an automatic analyzer which enables easy cleaning of cell contamination (reaction products and the like) of the automatic analyzer for a long period of time. It relates to reagents (particularly reagents for measuring urea nitrogen (UN) in blood (serum) and urine).

<Conventional Technology> Measurement of urea nitrogen (UN) in blood (serum) and urine is one of the most important in the field of clinical chemistry tests. Therefore, an automatic analyzer that has a large number of samples and can quickly process a large amount of samples without obtaining it is widely used as a daily analyzer (apparatus).

The basic configuration of the automatic analyzer is as follows (a) to (b) with respect to a number of cells (made of hard glass) arranged intermittently on a turntable in a circle and in a row and intermittently rotating. The operation (processing step) of (c) is sequentially performed to perform the operation continuously.

(A) Injection of sample (serum, urine, etc.) and reagent into cell and holding for a certain period of time (b) Spectroscopic measurement (c) Elimination of reaction solution in cell and washing of cell with pure water (3 times) The operation (step) such as (c) removing the reaction solution, injecting pure water, and removing the washing solution is performed in the case of a normal automatic analyzer in which the injection / exclusion nozzle is in a hard glass cell. This is performed by being inserted into.

<Problems to be Solved by the Invention> However, the cleaning of the inside of the cell is not sufficient with this level of cleaning, and during the use for a long period of time, dirt gradually adheres to or remains on the inner wall of the cell (especially ammonium). (Ions remain in the cell.) The continuous use for about 3 months deteriorates the analysis accuracy, and there is a drawback that the accuracy control becomes a serious problem (see FIG. 1, FIG. 1 will be described later (R1),
(R2), and UN in serum was measured by an automatic analyzer using the same cell, and the accuracy was expressed as a monthly coefficient of variation (CV).

The present inventors have pursued in more detail the cause of the deterioration of the analysis accuracy, and found that an electron microscope (electron microscope) generated on the inner wall of the cell (or existing from the beginning) due to repeated use of the cell.
Soil adheres to the target flaw (the phenomenon that this scratch occurs is called pitting), and it has been found to be a drag phenomenon based on the stain.

That is, extremely fine electron microscopic flaws (about several hundred angstroms in diameter) are generated (or exist from the beginning) on the inner wall of the cell, and reaction products in the reaction solution (probably ammonium ions) are present in the flaws. It was presumed that the particles adhered (or adsorbed) and could not be removed from the inside of the scratches by ordinary washing with pure water.

Therefore, the present inventors have eagerly developed a means for removing dirt in extremely fine electron microscope flaws generated on the inner wall of the cell (and existing from the beginning) with a predetermined number of cleanings of about three times with pure water. As a result of research, if a small amount of alkali metal salt is added in advance to the urea nitrogen measurement reagent used for analysis, dragging phenomenon will occur even when cells are used continuously for a long time (the effect of the previous measurement remains when cells are used continuously). The present invention has been found that the analysis accuracy is good and constant.

The reason that the addition of a small amount of alkali metal ions does not degrade the analysis accuracy in long-term continuous use and keeps it good and constant is that the alkali metal ions are a substitute for dirt (reaction products such as ammonium ions). ,
It is also considered to be due to effects such as salt dissolution on the adsorbed dirt.

<Means for Solving the Problem> The present application is composed of the following three claims (1) to (3).

(1) Ammonium ion remains in the cell of the automatic analyzer in a reagent for measuring urea nitrogen with an automatic analyzer by further reacting GLDH on ammonia released by the action of urease on the sample. A urea nitrogen measuring reagent for an automatic analyzer, characterized by coexisting an alkali metal salt in order to prevent urea.

(2) The reagent for measuring urea nitrogen for an automatic analyzer according to claim 1, wherein the alkali metal salt is potassium chloride.

(3) The reagent for measuring urea nitrogen for an automatic analyzer according to claim 1, wherein the alkali metal salt is sodium chloride.

The reagent for measuring urea nitrogen for an automatic analyzer according to the present invention refers to a reagent used for performing a urease-GLDH method for quantifying ammonia released by the action of urease.

The alkali metal salt used in the present invention mainly refers to potassium chloride, sodium chloride, lithium chloride, etc. (alkali metal chloride), but is not limited thereto, and inorganic salts that dissolve in water to generate ions of potassium, sodium, lithium, etc. Also, organic salts can be used at a concentration that does not inhibit the oxygen reaction such as urease or GLDH, and does not inhibit the oxygen reaction.

<Example 1> Urea nitrogen measuring reagents (R1) and (R2) for an automatic analyzer by the urease-GLDH method having the following composition were prepared (reagent compositions conventionally used).

(R1) Tris-HCl buffer (pH 7.3) 150 mM EDTA-2Na salt 0.5 mM Urease 10 U / ml (R2) Tris-HCl buffer (pH 7.3) 150 mM α-KG (ketoglutaric acid) 30 mM ADP 3 mM GLDH ( (Glutamate dehydrogenase, derived from bovine liver) 7U / ml β-NADH 0.6mM Next, the same reagent composition as (R1) and (R2) above was prepared by adding 0.3M sodium chloride to (R3) and (R4). The serum was used as a sample, and UN was measured by an automatic analyzer (Hitachi 736 automatic analyzer, washing with pure water three times). The result is shown in FIG.

According to the results of FIG. 2, the reagent composition (R
3), (R4) and reagent composition without salt (R1), (R2)
In comparison with the results analyzed by using FIG. 2, it can be seen that FIG. 2 (b) in which salt was added gave more stable results than FIG. 2 (a) in which salt was not added.

That is, in FIG. 2A, compared to the second lap,
The reason why the first lap shows a low value is that water is actually analyzed before this serum sample analysis, and dragging at the time of water measurement affects a subsequent sample (serum), resulting in a lower value. It is a thing. The dragging appears particularly as a difference in accuracy from the variation in the first lap, and is improved in the second and subsequent laps (FIG. 2 (a)). This is presumed to be because the reaction products (ammonium ions and the like) for each cell (each measurement) were not removed by electron microscopic flaws generated in the cell.

In FIG. 2, one round and two rounds indicate the number of rotations (the number of cells in one round) of the turntable of the automatic analyzer, and indicate the number of continuous use of the hard glass cell.

<Example 2> The following chlorides were added to the reagents (R1) and (R2) of Example 1 at different concentrations, and UN 160 mg / dl was obtained using each reagent composition.
The degree of drag from the sample to a pure water sample (UN 0 mg / dl) was investigated using a Hitachi 736 automatic analyzer. FIG. 3 shows the results.

(B) Potassium chloride (b) Salt (c) Lithium chloride (d) Magnesium chloride (e) Calcium chloride According to the results in FIG. 3, although the alkaline earth metal salt was not effective, the alkali metal The salt had a remarkable drag-preventing effect. Among them, it can be seen that the maximum effect is obtained with the minimum amount of KCl added. The reason is the solubility of ammonium ion (substituted ammonium ion) salt,
And the crystal structure is very similar to that of the salt of potassium ion. Therefore, it is presumed that the behavior of the cell against electron microscopic flaws is also similar, and that potassium ion substitutes for ammonium ion. It was thought to fulfill.

<Example 3> A reagent having a different salt concentration was prepared from the reagents (R1) and (R2) of Example 1, and a cell having a different degree of flaw in the cell was set using the reagent, and UN 160 mg was set. / dl from pure water sample (UN
(0 mg / dl) was investigated by Hitachi 736 automatic analyzer. The result is shown in FIG.

(A) Normally processed cell made of hard glass (normally a commercially available cell having electron microscopic irregularities on the inner wall surface) (b) Particularly hard glass cell whose inner wall surface is polished smoothly (electrode on the inner wall surface (C) Cell that has almost no visible irregularities (c) Cell using (b) for 3 months (cell in which electron microscopic irregularities are regenerated on the inner wall surface due to long-term use) According to the results in FIG. The cell (b) specially prepared shows good results at the beginning of use, but the cell (c) using the cell for 3 months showed almost the same drag as the ordinary cell (a). This phenomenon has an inherent problem in the durability of the cell material, even in the case of a polished cell. Electron microscopic flaws are generated on the inner surface of the cell by repeated use of the cell cleaning operation. It was presumed that the product was more likely to adhere, and as a result, the cell washing effect was deteriorated, resulting in a decrease in analysis accuracy.

<Effect of the Invention> Since the present invention is configured as described above, the cell cleaning effect of the automatic analyzer can be improved by adding a small amount of an alkali metal salt to the urea nitrogen (UN) measuring reagent for the automatic analyzer. This has the effect that measurement can be performed accurately for a long time without the need for cell replacement due to the occurrence of scratches.

[Brief description of the drawings]

FIG. 1 is a graph showing the monthly variation coefficient (CV) of the UN measurement value in serum. FIG. 2 shows the results for the case where no salt was added (a) and the case where salt was added (b).
5 is a graph showing the effect of an alkali metal salt using a UN measurement reagent as viewed from UN measurement values. FIG. 3 is a graph in which the most effective alkali metal among the alkali metal salts was examined from the degree of drag using the UN measurement reagent to which various alkali metal salts were added. FIG. 4 is a graph in which a cell in which the state of the inner wall of the cell is changed is set, and the cell difference is observed from the dragging degree of the cell.

Claims (3)

(57) [Claims] 1. An ammonium ion remaining in a cell of an automatic analyzer in a reagent for measuring urea nitrogen by an automatic analyzer by further reacting GLDH on ammonia released by the action of urease on a sample. To prevent
A urea nitrogen measuring reagent for an automatic analyzer, wherein an alkali metal salt coexists. 2. The reagent for measuring urea nitrogen for an automatic analyzer according to claim 1, wherein the alkali metal salt is potassium chloride. 3. The reagent for measuring urea nitrogen for an automatic analyzer according to claim 1, wherein the alkali metal salt is sodium chloride.