JP2004135660A - Method for measuring bio-component having antiseptic resistance and reagent for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring neutral fat having high antiseptic resistance and no reduction in various functions such as no generation of essential turbidity during storage or magnesium, especially used for clinical diagnosis, and obtain a reagent for the same. <P>SOLUTION: The method for measuring the neutral fat is carried out so that both the effect of antiseptic performance and the suppression of performance reduction of the reagent are compatible continuously during storage in the presence of the antiseptic. <P>COPYRIGHT: (C)2004,JPO

Description

(4) The present invention relates to a method for measuring neutral fat or magnesium and a reagent thereof. In particular, the present invention relates to a reagent for measuring neutral fat or a reagent for measuring magnesium in a biological component used for clinical diagnosis.

診断 As a diagnostic agent used in the clinical test field, there is a biochemical measurement reagent utilizing an enzyme reaction. In this reagent, since the enzyme is unstable in the solution, the enzyme and unstable substances other than the enzyme were freeze-dried and dissolved in a lysis solution before use. For this reason, there is a problem that since the freeze-drying is performed on the manufacturing side, the cost and the manufacturing time are increased, and the dissolution is troublesome on the user side. On the other hand, in recent years, unprepared liquid reagents that do not need to be prepared have been developed in the field of biochemical testing, and the problems associated with freeze-drying have been solved. This liquid reagent has been completed as a reagent that can be stored in a solution state for a long period of time by stabilizing the raw enzyme, coenzyme, etc. by various methods. I have.

In the development of unprepared liquid reagents, one of the development points to be newly added from the conventional freeze-dried reagents is to improve the preservative ability during distribution. Various additives have been studied to improve the preservative ability, and the results have been reported (for example, see Patent Document 1). However, in the selection of preservatives, while taking into consideration preservative performance such as antibacterial activity and the long-term stability of the preservative itself, on the other hand, the influence on the stability of raw material enzymes and coenzymes cannot be ignored, In practice, it has been found that preservatives may deteriorate component enzymes and the like due to compatibility between preservatives and enzymes, and may have a considerable effect on reagent performance such as precision and accuracy. As described above, it is a very difficult task to achieve both sufficient preservative performance and reagent stability.
Tokuhei 7-78005

As reagents whose performance of the measuring reagent is deteriorated by the preservative, there are a neutral fat measuring reagent and a magnesium measuring reagent (for example, see Patent Documents 2 and 3). The measurement of neutral fat is generally performed by measuring glycerophosphate or ADP generated by allowing lipase and glycerol kinase to act on neutral fat. Above all, a method has been widely used in which hydrogen peroxide generated by reacting glycerophosphate oxidase with glycerophosphate acts on a chromogen under peroxidase to form a quinone dye. In addition, when measurement is performed in a two-reagent system using an automatic analyzer or the like, ascorbate oxidase is added to the first reagent, and ascorbic acid, which is a substance that interferes with the measurement, is often removed in the art. It is. Also, the addition of catalase to eliminate endogenous glycerol is well performed in the art.
The reagent for measuring magnesium is generally measured by measuring glycerophosphate or ADP which generates glycerol kinase activity activated by magnesium.
Japanese Patent Publication No. 58-5677 JP-A-64-30597

複数 A plurality of enzymes are used in these measurement reagents. Some enzymes are relatively insensitive to preservatives such as peroxidase, while others are greatly affected by glycerol kinase. In a group of reagents called biochemical diagnostic reagents such as neutral fat measurement reagents, peroxidase is rarely used alone as an enzyme and is used in combination with other enzymes in a multi-step reaction, so it is used as a preservative as a reagent Drug resistance depends on the least resistant enzyme. Therefore, even if peroxidase alone has high antiseptic resistance, it is not useful as a reagent.

The problem to be solved by the present invention is to provide a neutral fat measuring reagent and a magnesium measuring reagent which have high resistance to preservatives and maintain the precision and accuracy of the measuring reagent for a long period of time. is there.

The present inventors have studied the interaction between components such as enzymes and dyes in the reagents and preservatives to achieve the above object, and as a result of intensive studies, selected an appropriate combination of enzyme and preservative. As a result, the component enzymes in the reagent are degraded by the preservative, causing turbidity during storage in the neutral fat measurement reagent, increasing the reagent blank during the reaction, and decreasing the ability to erase endogenous free glycerol The lowering of the high-value measurement limit causes turbidity during storage of the magnesium measurement reagent, the increase of the reagent blank during the reaction, the decrease in sensitivity, and the reduction of the high-value measurement limit results in the precision of the measurement reagent. The present inventors have found that the properties and accuracy have been reduced, and have completed the present invention. That is, the present invention has the following configuration.
(1) A method for measuring neutral fat, which has high resistance to preservatives and does not substantially cause turbidity during storage (2) High resistance to preservatives, and Neutral fat measurement method characterized by substantially no increase in reagent blank during storage (3) High resistance to preservatives and no substantial decrease in glycerol scavenging ability during storage (4) Neutral fat measurement method characterized in that it has high resistance to preservatives and that the high value measurement limit does not substantially decrease during storage (5) Preservation (6) a method for measuring magnesium characterized in that it has a high resistance to preservatives and that substantially no turbidity occurs during storage; Magnesium measurement characterized by no rise in reagent blank Method (7) A method for measuring magnesium, which has high resistance to preservatives and does not substantially reduce sensitivity during storage (8) High resistance to preservatives and storage A neutral fat measuring reagent characterized in that substantially no turbidity is generated therein (9) having a high resistance to preservatives and substantially no rise of a reagent blank during storage; Neutral fat measuring reagent (10) which has high resistance to preservatives and which does not substantially reduce glycerol scavenging ability during storage (11) (12) a neutral fat measuring reagent characterized in that it has high resistance to preservatives, and has substantially no lowering limit during storage. Magnesium measurement characterized by no turbidity Reagent (13) a magnesium measurement reagent characterized by having a high resistance to preservatives and substantially no rise of the reagent blank during storage; (14) having a high resistance to preservatives; And a reagent for measuring magnesium, wherein the sensitivity does not substantially decrease during storage.

In other words, the present invention has the following configuration.
[1]
A method for measuring neutral fats, characterized in that the effect of preservative performance and suppression of reagent performance deterioration are compatible at the same time during storage in the presence of a preservative [2].
The method of [1], wherein suppression of reagent performance deterioration is at least one selected from the following: (1) substantially no turbidity; (2) substantially no increase in reagent blank; (4) Substantially no lowering of the high value measurement limit (5) Substantially no reduction in enzyme activity contained in the reagent [3]
The method for measuring magnesium, characterized in that the effect of preservative performance and the suppression of reagent performance deterioration are compatible at the same time during storage in the presence of a preservative [4].
The method of [3], wherein the suppression of reagent performance reduction is at least one selected from the following: (1) substantially no increase in reagent blank (2) substantially no reduction in sensitivity (3) substantial That the enzyme activity contained in the reagent does not decrease [5]
The method [6] according to any one of [1] to [4], wherein the preservative is a preservative that acts directly on the protein.
In the presence of a preservative, a neutral fat measuring reagent [7] characterized in that the preservative performance effect and the suppression of reagent performance deterioration are both compatible during storage.
In the presence of a preservative, a reagent for measuring magnesium, characterized in that the effect of preservative performance and the suppression of reagent performance deterioration are compatible at the same time during storage.

According to the present invention, it is possible to provide a neutral fat measuring reagent and a magnesium measuring reagent which have high resistance to preservatives and maintain the precision and accuracy of the measuring reagent for a long period of time.

One embodiment of the present invention is a neutral fat measurement reagent comprising a two-reagent system containing glycerol kinase, glycerophosphate oxidase, and the like as a first reagent, and lipase, peroxidase, and the like as a second reagent. This is a method for measuring neutral fats, which has high resistance to water and does not generate turbidity during storage.

When an antibiotic is used as a preservative, microorganisms can relatively easily suppress the membrane permeability of the substance or acquire the ability to decompose and detoxify the substance, thereby reducing the occurrence of resistant bacteria. It is often thought that the preservative effect of the reagent is substantially lost. On the other hand, preservatives that can act directly on proteins are more preferable in that it is considered difficult for microorganisms to obtain resistance, and are likely to be widely used in the future. However, preservatives that directly act on such proteins naturally act on coexisting enzyme proteins, and may cause instability depending on the protein structure. Thus, preservative resistance is considered to be due to both the mechanism of action of the preservative and the structure of the protein.

Such a preservative is not particularly limited, but is isothiazoline such as 2-methyl-4-isothiazolin-3-one-hydrochloride (also referred to as N-methylisothiazolone (N-methylisothiazolone; abbreviated as MIT)). Compound, 2-hydroxypyridine-N-oxide (abbreviation: HPO), chloracetamide (abbreviation: CAA), {N, N-methylene-bis [(N-1-hydroxymethyl) -2,5-dioxo-4-imidazolidinyl] ] -Urea (abbreviation IZU) and 5-bromo-5-nitro-1,3-dioxane (abbreviation BND).

When using these, it is preferable to use as few types as possible. More preferably, one type is used. The reason is that a measurement system that follows a multi-step reaction, such as a neutral fat measurement reagent or magnesium measurement reagent, requires many components such as enzymes and dyes, but the effect of preservatives on each component is different. This is because the number of elements affected is considered to be as small as possible because it is considered to differ depending on the preservative.

For example, in the case of a neutral fat measurement reagent, glycerol kinase may be damaged if BND or MIT coexists. In addition, coexistence of IZU may damage glycerophosphate oxidase. In addition, ascorbate oxidase may be damaged when HPO coexists. In addition, coexistence of CAA may affect the time course of the color reaction, which is considered to be due to deterioration of 4-aminoantipyrine. Therefore, for example, when selecting an appropriate preservative from among the above-mentioned MIT, HPO, CAA, IZU and BND, if multiple preservatives are used, it is necessary to deal with the influence on many factors, but only one kind is required. If used, only one element needs to be addressed. For example, when using MIT, it is only necessary to deal with glycerol kinase. The same applies to other preservatives.

特 に Particularly preferred preservatives in the present invention include isothiazoline compounds which are said to act on SH groups and amino groups of proteins. For example, 2-methyl-4-isothiazolin-3-one (or N-methylisothiazolone (abbreviated as MIT)), 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 1, Examples include 2-benzisothiazolin-3-one (BIT) and derivatives thereof. These are Proculin 150 (manufactured by Spelco), Proculin 300 (manufactured by Spelco), Activide MBS (manufactured by So Japan) as a mixture, Activide B20 (N) (manufactured by So Japan) as a single product, and MIT (manufactured by Roche) And Sigma) are commercially available. In addition, an embodiment in which a preservative other than the isothiazoline-based compound is not added to the reagent is more preferable for the reasons described above. In the method for measuring neutral fat according to the present invention, the isothiazoline-based compound is excellent in both the preservative ability and the effect on the stability of the reagent.

防 The preservative used in the present invention refers to a substance added to a reagent for the purpose of suppressing the growth of microorganisms during storage of the reagent. At this time, the concentration of the preservative is not particularly limited, but is desirably a concentration at which a sufficient preservative performance effect is obtained. It goes without saying that the concentration of the preservative varies depending on the kind of the preservative, the composition of the reagent to be added, and the like, and a person skilled in the art can appropriately determine the appropriate concentration to be added. The concentration is not particularly limited as long as it has a sufficient preservative ability and is in a concentration range used for diagnostic use. For example, the lower limit concentration in the solution is 0.0001%, preferably 0.001%, more preferably 0.01%. is there. The upper limit concentration is 1%, preferably 0.5%, more preferably 0.1%.

Sufficient preservative performance effect can be seen, for example, by the following antibacterial test, with no bacterial growth. In the antibacterial test method, a measurement reagent to which a preservative is added is used as a test reagent, and this is stored at 35 ° C. for one week or two weeks. Brain Heart Infusion (manufactured by Difco Laboratories) is added to and mixed with this reagent to a final concentration of 39 g / L to prepare a test reagent. Bacterial bacterium is a bacterium such as Pseudomonas sp., Stenotrophomonas sp., Enterobacter sp., Proteus sp., Klebsiella sp., Escherichia sp., Bacillus sp., Criptococcus sp., Candida sp. The suspension is prepared so that the absorbance at 660 nm becomes 0.5 Abs as a standard. After inoculating 50 μL of the bacterial solution with 4 mL of the test reagent, the mixture is shaken at 25 or 37 ° C. for 3 days. Judgment was made by comparing the absorbance of the test reagent at 660 nm before and after storage. If there was a significant increase in absorbance, it was judged that there was no preservative effect. It is determined that there is an effect.

高 い High resistance to preservatives means that, in the presence of preservatives, the preservative performance effect and the suppression of reagent performance degradation are both compatible during storage. For example, a preservative whose resistance is to be tested is added to a certain composition, and during storage, a decrease in reagent performance such as accuracy, precision, and measurement range is substantially suppressed, and a suspended substance or a precipitate is substantially prevented. If the generation of substances is suppressed, it can be said that the reagent has high resistance to the preservative. In this definition, the storage temperature and the storage period can be arbitrarily set within a range of common knowledge of those skilled in the art. Usually, it is preferably performed at 2 to 10 ° C. for 0.5 years or more, preferably 1 year or more, more preferably 1.5 years or more, or at room temperature (around 25 ° C.) for 0.5 years or more. When the test is performed, the test can be performed at 35 to 37 ° C. for one week or more as an accelerated test. In that case, the conditions and period of the test can be changed as necessary.

Due to the decrease in reagent performance in the presence of preservatives, turbidity occurs during storage, the reagent blank rises during the reaction, the ability to eliminate endogenous free glycerol decreases, and The lower limit is seen as a phenomenon. In the case of the magnesium measurement reagent, phenomena such as turbidity during storage, an increase in the reagent blank during the reaction, a decrease in sensitivity, and a decrease in the high value measurement limit are observed. Whether or not the decrease in reagent performance among the resistance to preservatives is suppressed can be evaluated by observing the transition of these phenomena.

These phenomena are thought to be caused by deterioration of various elements such as enzymes and dyes. Decrease in the elimination ability of endogenous free glycerol and decrease in the measurement limit of high value are mainly caused by slowing down the follow-up of at least one of the multi-step enzyme reactions due to inactivation of the enzyme. Responsible. In the neutral fat measurement reagent, glycerol kinase, glycerophosphate oxidase, and catalase are exemplified as enzymes involved in glycerol elimination. In addition, examples of the enzymes involved in the measurement include lipase, glycerol kinase, glycerophosphate oxidase, and peroxidase. Turbidity during storage and rise in reagent blank during the reaction are mainly caused by denaturation and insolubilization of the enzyme protein due to deactivation.In the case of turbidity, other substances such as surfactants and salts are further added. In some cases, it may be insolubilized by the action of a preservative, and in the case of a blank rise, the dyes may be self-condensed by the action of a preservative.

Conventionally, when an isothiazoline-based compound is used as a preservative, it is thought that glycerol kinase is mainly brought about by deterioration by the preservative. Therefore, for example, when an isothiazoline-based compound is used as a preservative in the present invention, whether or not the decrease in reagent performance among the preservatives is suppressed depends on the concentration of the preservative of glycerol kinase having a concentration of 0.1 to 20 U / mL. It can also be evaluated by observing the deterioration due to. Whether or not the decrease in reagent performance is suppressed is determined based on whether or not "enzyme activity contained in the reagent does not substantially decrease". The glycerol kinase activity residual rate when coexisting at a preservative concentration of 100 mg / L at 25 ° C. for one week is 70% or more, preferably 80% or more, and more preferably 90% or more. When it is desired to evaluate the effect on a specific preservative, the preservative at the above concentration may be added to the reagent composition to be evaluated, and the enzyme activities before and after storage under the above conditions may be compared.

As described above, depending on the type of preservative to be used, the effect on the enzyme differs depending on the type of preservative. It can be selected and evaluated similarly to glycerol kinase. The enzyme serving as an indicator is not particularly limited, but lipase, glycerophosphate oxidase, ascorbate oxidase, and the like can be preferably selected as the neutral fat measurement reagent. Whether or not the decrease in reagent performance is suppressed is determined by determining whether the residual ratio of the enzyme activity when coexisting at 25 ° C. for 1 week at a concentration of 100 mg / L of the preservative is 70% or more, preferably 80% or more, more preferably 90% or more. It is. The same applies to the magnesium measurement reagent.

に お い て In the present invention, “substantially no turbidity” means that no turbidity is generated or almost no turbidity is generated and the turbidity is suppressed to a certain level or less. The “constant level” is appropriately determined by a person skilled in the art, but in general, any level that satisfies the standard of the reagent (product standard, shipping standard, etc.) may be used. The turbidity during storage can be evaluated by visual observation, measurement of absorbance at 660 nm, or measurement by a particle counter (manufactured by Kurabo Industries, Ltd.). Among them, a particle counter is preferable from the viewpoint of minimum detection sensitivity. The total count of 5 to 150 μm in the particle counter is 1000 / mL or less, preferably 500 / mL or less, and more preferably 100 / mL or less. For the determination of the measurement result, any conventional test method such as an evaluation method used in the field of quantitative inspection or analytical chemistry can be appropriately adopted.

One embodiment of the present invention is a neutral fat measurement reagent comprising a two-reagent system containing glycerol kinase, glycerophosphate oxidase, and the like as a first reagent, and lipase, peroxidase, and the like as a second reagent. This is a method for measuring neutral fat, which has high resistance to lipase and does not raise a reagent blank during storage.

試 薬 In the present invention, the reagent blank refers to the absorbance measured at the measurement wavelength at the end of measurement when measuring a sample containing no purified water or the object to be measured. "Substantially no increase in the reagent blank" means that there is no rise or no increase in the blank at all, and the blank is suppressed to a certain level or less. In the present invention, the rise after storage of the reagent blank relative to before storage is 0.1 Abs or less, preferably 0.05 Abs or less, more preferably 0.01 Abs or less. For the determination of the measurement result, any conventional test method such as an evaluation method used in the field of quantitative inspection or analytical chemistry can be appropriately adopted.

One embodiment of the present invention is a neutral fat measurement reagent comprising a two-reagent system containing glycerol kinase, glycerophosphate oxidase, and the like as a first reagent, and lipase, peroxidase, and the like as a second reagent. This is a method for measuring neutral fat, which has high resistance to glycerol and does not decrease glycerol scavenging ability during storage.

In the present invention, the ability to eliminate glycerol is an ability to convert (eliminate) free glycerol, which is a hindrance when contained in a sample in the measurement of neutral fat, into a substance that does not participate in the reaction before the measurement of neutral fat. Means The expression "the glycerol scavenging ability does not substantially decrease" means that the scavenging ability does not decrease at all or hardly decreases and is suppressed to a certain level or more. The “constant level” is appropriately determined according to the common knowledge of those skilled in the art, but generally may be any level that satisfies the reagent specifications. Usually, 100 mg / dL in terms of triolein is sufficient, but is preferably 2000 mg / dL or more, and more preferably 3500 mg / dL or more. Under these conditions, the decrease in glycerol-erasing ability of the present invention after storage as compared to before storage is 30% or less, preferably 20% or less, more preferably 10% or less.

The glycerol scavenging ability is preferably determined as follows.
(1) Glycerol (manufactured by Nacalai Tesque, Inc.) is diluted with purified water so that the concentration becomes equal, and a dilution series of 10 levels or more is prepared.
(2) The above sample is measured twice or more each using a neutral fat measurement reagent, and the average value is calculated. The measurement uses a Hitachi 7170 type automatic analyzer. 180 μL of the first reagent is added to 2.1 μL of the sample, and incubated at 37 ° C. for 5 minutes to obtain a first reaction. Thereafter, 90 μL of the second reagent is added and incubated for 5 minutes to obtain a second reaction. The absorbance at 600 nm is measured by a two-point end method by taking the difference between the absorbances of the first reaction and the second reaction after correcting the liquid volume. The absorbance measurement result is calculated from the measured absorbance of purified water and an aqueous solution of 200 mg / dL triolein, and is determined as a neutral fat concentration.
(3) If completely erased, the measured value should be zero. Plot the measured levels of triolein at each level to see how far they are at zero. If the measured value exceeds 10 mg / dL (5% of 200 mg / dL triolein), it is determined that there is no glycerol scavenging ability at higher concentrations including this concentration level, and the concentration below one level is regarded as the glycerol scavenging ability. I do.

One embodiment of the present invention is a neutral fat measurement reagent comprising a two-reagent system containing glycerol kinase, glycerophosphate oxidase, and the like as a first reagent, and lipase, peroxidase, and the like as a second reagent. This is a method for measuring neutral fats, which has high resistance to lipase and does not lower the high value measurement limit.

In the present invention, the high value measurement limit refers to the highest value of the concentration at which the measured value is proportional to the dilution series when a sample obtained by serially diluting a neutral fat high value sample is measured.
The phrase "substantially does not lower the high measurement limit" means that the measurement limit does not decrease at all or hardly decreases, and is suppressed to a certain level or more. The “constant level” is appropriately determined according to the common knowledge of those skilled in the art, but generally may be any level that satisfies the reagent specifications. Usually, 1500 mg / dL in terms of triolein is sufficient, but it is preferably 2000 mg / dL or more, and more preferably 2500 mg / dL or more. Under these conditions, the reduction of the high measurement limit in the present invention after storage relative to before storage is 30% or less, preferably 20% or less, more preferably 10% or less.

The high measurement limit is preferably determined as follows.
(1) Triolein (manufactured by Nacalai Tesque, Inc.) is diluted with purified water so that the concentration becomes equal, to prepare a dilution series of 10 levels or more.
(2) The above sample is measured twice or more each using a neutral fat measurement reagent, and the average value is calculated. The measurement uses a Hitachi 7170 type automatic analyzer. 180 μL of the first reagent is added to 2.1 μL of the sample, and incubated at 37 ° C. for 5 minutes to obtain a first reaction. Thereafter, 90 μL of the second reagent is added and incubated for 5 minutes to obtain a second reaction. The absorbance at 600 nm is measured by a two-point end method by taking the difference between the absorbances of the first reaction and the second reaction after correcting the liquid volume. The absorbance measurement result is calculated from the measured absorbance of purified water and an aqueous solution of 200 mg / dL triolein, and is determined as a neutral fat concentration.
(3) Plot the value obtained by dividing the measured value of triolein at each level by the measured value of the first level to see how far the proportional relationship is maintained. If the value deviates from ± 5% of the theoretical value, it is determined that there is no linearity at a concentration higher than this concentration level, and a concentration one level lower is defined as a high value measurement limit.

One embodiment of the present invention is a magnesium measuring reagent comprising a two-reagent system containing glycerophosphate oxidase or the like as a first reagent and glycerol kinase or glycerol as a second reagent, and has a high antiseptic property. A magnesium measuring reagent that is resistant and does not generate turbidity during storage.

One embodiment of the present invention is a magnesium measuring reagent comprising a two-reagent system containing glycerophosphate oxidase or the like as a first reagent and glycerol kinase or glycerol as a second reagent, and has a high antiseptic property. A magnesium measuring reagent that is resistant and does not raise the reagent blank during storage.

上昇 In the present invention, the increase of the reagent blank after storage with respect to before storage is 0.1 Abs or less, preferably 0.05 Abs or less, more preferably 0.01 Abs or less.

One embodiment of the present invention is a magnesium measuring reagent comprising a two-reagent system containing glycerophosphate oxidase or the like as a first reagent and glycerol kinase or glycerol as a second reagent, and has a high antiseptic property. A magnesium measuring reagent that is resistant and does not decrease in sensitivity during storage.

In the present invention, the sensitivity refers to an absorbance obtained by subtracting a reagent blank absorbance from an absorbance when a sample having a known concentration in a range in which quantitativeness is guaranteed is measured.
The phrase "substantially does not lower the sensitivity" means that the sensitivity does not decrease at all or hardly decreases and is suppressed to a certain level or more. The “constant level” is appropriately determined according to the common knowledge of those skilled in the art, but generally may be any level that satisfies the reagent specifications. Under these conditions, the decrease in sensitivity in the present invention after storage as compared to before storage is 30% or less, preferably 20% or less, more preferably 10% or less.

Further, one embodiment of the present invention is a method for measuring neutral fat comprising a two-reagent system containing glycerol kinase, glycerophosphate oxidase or the like as a first reagent, and lipase or peroxidase as a second reagent. This is a method for measuring neutral fats that has high resistance to preservatives and further satisfies at least two of the following [1] to [4]. [1] Substantially no turbidity during storage, [2] Substantially no rise in reagent blank during storage, [3] Substantially no decrease in glycerol erasing ability during storage, [4] Storage During the time the high measurement limit is not substantially reduced.

One embodiment of the present invention is a magnesium measuring reagent comprising a two-reagent system containing glycerophosphate oxidase or the like as a first reagent and glycerol kinase or glycerol as a second reagent, and has a high antiseptic property. This is a method for measuring magnesium that has resistance and further satisfies two or more of the following [1] to [3]. [1] Substantially no turbidity during storage, [2] Substantially no increase in reagent blank during storage, [3] Substantially no decrease in sensitivity during storage.

The glycerol kinase used in the present invention includes an enzyme that catalyzes the following reaction classified into EC 2.7.1.30.
Glycerol + ATP + Mg → Glycerol-3-phosphate + ADP + Mg
The glycerol kinase used in the present invention is preferably one having a preservative resistance of 70 mg / L and a residual glycerol kinase activity of 70% to 100%, preferably 80% when coexisted at 25 ° C. for 1 week at a concentration of 100 mg / L of the preservative. % To 100%, more preferably 90% to 100%.

起源 The origin of such an enzyme is not particularly limited. If it is of animal origin, human, rat liver, pigeon liver and the like are exemplified. Microorganisms such as yeasts and microorganisms include Saccharomyces (for example, Saccharomyces cerevisiae), Candida (for example, Candida mycoderma), and Bacillus (for example, Bacillus subtilis). ), Bacillus stearothermophilus, the genus Thermus (e.g., Thermus flavos), the genus Cellulomonas (e.g., Cellulomonas? Flavigena, Cellulomonas esulomonas. Escherichia (eg Escherichia coli, Arthrobacter sp. (Eg, Arthrobacter sp.), Flavobacterium sp. (Eg, Flavobacterium meningosepticum), Streptomyces The genus Ases (eg, Streptomyces canus), Geotricum candydam (Geotricum candidum), and the like. Any of these can be used commercially available products.

Of these, those that are preferably used in that they maintain the stability of the reagent even when coexisting with preservatives, particularly preservatives that directly act on proteins, are of the genus Cellulomonas and Flavobacterium (Flavobacterium medicum). Ningosepticum), the genus Arthrobacter (Esp. Sp.), The genus Thermus, and the genus Bacillus (Bacillus stearothermophilus). More preferably, they belong to the genus Cellulomonas, Thermus and Bacillus (Bacillus stearothermophilus). Most preferably, those extracted from Cellulomonas sp. The above enzymes include those produced from genetically modified microorganisms in which these genes have been incorporated into other microorganisms.

One of the reasons why such a difference occurs is considered to be due to the number and position of moieties susceptible to reaction, for example, sulfur-containing amino acid residues such as cysteine and methionine possessed by the enzyme protein (for example, For the amino acid sequence of glycerol kinase derived from Thermus flavus, see Patent Document 4). As a suggestion of this, there is a case where sarcosine oxidase achieves stabilization against various substances by substituting a cysteine residue with another amino acid (for example, see Patent Document 5). Therefore, the effect of chemicals such as preservatives may be affected when enzymes containing sulfur-containing amino acids are present, especially when sulfur-containing amino acids are present on the enzyme surface, especially when the sulfur-containing amino acids are present near the active center, coenzyme or substrate binding site. It is suggested that they are more susceptible. In addition, it is considered that the smaller the number of sulfur-containing amino acids is, in principle, the better the preservative resistance unless there is a large difference in other conditions.
JP-A-11-9279 Japanese Patent No. 3132618

下限 The lower limit of the concentration of the glycerol kinase is usually 0.05 U / mL, preferably 0.1 U / mL. On the other hand, the upper limit is usually 20 U / mL, preferably 5 U / mL, and more preferably 2 U / mL.

緩衝 The buffer used in the present invention includes Tris buffer, phosphate buffer, GOOD buffer and the like. Above all, the pH of the Tris buffer solution and the phosphate buffer solution easily varies depending on the concentration and temperature, but has the advantage of being inexpensive. On the other hand, examples of GOOD buffers having small fluctuations in pH include MES, Bis-Tris, ACES, BES, MOPS, PIPES, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS. Any of these can be used commercially available products.

ＰＨ The pH of the present invention is not particularly limited as long as it does not destabilize the enzyme. The lower limit of the pH of the buffer is preferably 5, but more preferably 5.5. The upper pH limit is preferably 9, but is more preferably 8. In the case of a neutral fat measurement reagent, the pH 6 to 7.5, and more preferably a weakly acidic range of 6 to 7 is more preferable because the reaction pH of glycerophosphate oxidase and ascorbate oxidase is optimal.

界面 A surfactant, a saccharide, a chelating agent, a coenzyme, and the like may be further added to the measurement reagent of the present invention. Examples of the surfactant include a nonionic surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant. Examples of the saccharide include mannitol, sorbitol, cyclodextrin and derivatives thereof. Examples of the chelating agent include ethylenediaminetetraacetic acid and salts thereof. Examples of the coenzyme include flavin adenine dinucleotide. Any of these can be used commercially available products. The purpose of these additions is not particularly limited, but is preferably a type and concentration having a stabilizing effect on enzymes and reagents. Further, it is preferable that the additive itself be of a kind and concentration that does not undergo deterioration by the preservative.

測定 The measurement reagent of the present invention may contain other reagents necessary for measurement. The neutral fat measurement reagent may include, for example, lipase, glycerol kinase, glycerophosphate oxidase, ATP, magnesium, peroxidase, and a chromogen. Further, it may contain, for example, lipase, glycerol kinase, ATP, ADP-dependent hexokinase, glucose-6-phosphate dehydrogenase, and oxidized nicotinamide adenine dinucleotide. In addition, the magnesium measurement reagent may generally contain glycerol kinase, glycerophosphate oxidase, ATP, a chelating agent, peroxidase, and a chromogen.

Among the above various components, lipase, glycerol kinase, glycerophosphate oxidase, ATP, magnesium, peroxidase, a neutral fat measuring reagent using a chromogen, glycerol kinase, glycerophosphate oxidase, ATP, chelating agent, peroxidase, color With respect to a reagent for measuring magnesium using a source body, a particularly preferable effect is obtained by the present invention.

Further, in the present invention, a saccharide may be added. The saccharide is appropriately selected from monosaccharides, disaccharides, oligosaccharides, cyclic oligosaccharides and the like, and is not particularly limited. For example, xylose, glucose, galactose, fructose, sucrose, lactose, trehalose, maltose, 2-deoxy-D-glucose, melibiose, ribitose, inositol, dulcitol, glucitol, glucono-1,5-lactone, G2-β-cyclodextrin Sucrose monoca plate, sucrose monocholate and the like. Among them, compounds having sucrose as a skeleton, for example, sucrose, lactosucrose, raffinose, inulooligosaccharides, sucrose monocaprate, sucrose monocholate and the like are preferably used. Among them, sucrose is preferred. In each case, commercially available products can be used. The concentration of the added saccharide is not particularly limited, but is preferably 0.01 times (W / W) or more with respect to the preservative, and the lower limit is preferably 0.05 times (W / W). The upper limit is 500 times the amount (W / W). In particular, when an isothiazoline-based compound is used as a preservative, the resistance (stabilizing effect) increases, which is preferable.

The measurement of glycerol kinase activity is preferably performed under the following measurement conditions.
Using glycerol as a substrate, the enzyme activity was measured by the amount of glycerol-3-phosphate produced. 0.2 ml of 0.5% aqueous solution of 4-aminoantipyrine, 0.2 ml of 1.5% aqueous phenol solution, 200 U of glycerol-3-phosphate oxidase, 80 U of peroxidase, 48.4 mg of ATP, and 0.1 M HEPES buffer (pH 7.9) To make a total volume of 21 ml, which was used as a stock solution for the following measurement. In each reaction, 3 ml of this stock solution was taken, 50 μl of a 0.3 M glycerol aqueous solution and 100 μl of an enzyme solution were added, and after mixing, the absorbance at 500 nm was recorded for 3 minutes with a spectrophotometer controlled at 37 ° C. The change in absorbance per minute was determined from the portion (ΔODtest). In the blind test, 100 μl of an enzyme diluent (20 mM potassium phosphate buffer containing 0.2% bovine serum albumin, pH 7.5) was added in place of the enzyme solution, and the same procedure was followed to determine the change in absorbance per minute. (ΔODblank).
The enzyme activity of glycerol kinase was calculated from the obtained change in absorbance based on the following formula. The amount of the enzyme that phosphorylates 1 micromol of glycerol per minute under the above conditions is defined as 1 unit (1 U).
Calculation formula activity value (U / ml) = ｛ΔOD / min (ｍｉｎODtest-ｔｅODblank) × 3.15 (ml) × dilution factor｝ / ｛13.3 × 1/2 × 1.0 (cm) × 0.1 (Ml)｝
3.15 ml = reaction mixture liquid volume 13.3 = quinone dye extinction coefficient 1/2 under the above measurement conditions = 1/2 molecule of quinone dye formed from one molecule of hydrogen peroxide generated by enzyme reaction Coefficient 1.0 cm = cell optical path length 0.1 ml = enzyme sample volume

Hereinafter, the present invention will be described specifically with reference to examples. The present invention is not particularly limited by the examples.
(Example 1)
The first reagent for measuring neutral fats described below to which N-methylisothiazolone (manufactured by Roche) and glycerol kinase (GYK-301 manufactured by Toyobo Co., Ltd., derived from Cellulomonas sp.) Was stored at 25 ° C. for 1 week, and the remaining activity was determined. The ratio of the activity value after storage to the activity value immediately after dissolution) was examined. In Comparative Examples, glycerol kinase was derived from Cellulomonas sp. And derived from Thermos fullerbus (Toyobo Co., Ltd.).

(Preparation of reagent)
A first reagent of a neutral fat measuring reagent having the following composition was prepared.
First reagent PIPES-NaOH 50 mM pH 6.6
MgCl2 0.2g / L
Adenosine triphosphate 2Na salt 0.9 g / L
Emulgen A60 2g / L
Triton X-100 1g / L
4-aminoantipyrine 0.1 g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Glycerol kinase 3U / mL
Glycerophosphate oxidase (G3O-311 manufactured by Toyobo) 5 U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo) 3 U / mL
Catalase (Toyobo) 200U / mL
N-methylisothiazolone 100mg / L

Results shown in Table 1. In the comparative example, the activity was reduced to about 40%, whereas in the example, the stability was 90% or more even after storage.

(Example 2)
The following second reagent for measuring magnesium to which N-methylisothiazolone (manufactured by Roche) and glycerol kinase (GYK-301 manufactured by Toyobo Co., Ltd. derived from Cellulomonas sp.) Was stored at 25 ° C. for one week, and the remaining activity (immediately after dissolution) (The ratio of the activity value after storage to the activity value). In Comparative Examples, glycerol kinase was derived from Cellulomonas sp. And derived from Thermos fullerbus (Toyobo Co., Ltd.).

(Preparation of reagent)
A second reagent of a magnesium measuring reagent having the following composition was prepared.
Second reagent PIPES-NaOH 50 mM pH 6.8
Disodium ethylenediaminetetraacetate 3g / L
Adenosine diphosphate 2Na salt 0.5 g / L
4-aminoantipyrine 0.3 g / L
Glycerol kinase 6U / mL
Glycerin 0.4g / L
N-methylisothiazolone 100mg / L

Results shown in Table 2. In Comparative Examples, the activity was reduced to about 60%, whereas in Examples, good stability of 90% or more was exhibited even after storage.

(Example 3)
To the first test solution of the neutral fat measurement reagent of Example 1, the following reagents were combined as the second reagent, and the following measurement methods were used to dilute glycerol 5000 mg / dL (in terms of triolein) with each reagent at 10 levels. The glycerol scavenging ability and the high measurement limit were calculated, and the performance after storage at 25 ° C. for 1 week before storage was shown as a relative value (%). The glycerol scavenging ability was judged to be the highest concentration of the glycerol dilution level at which the measured value was 3 mg / dL or less as the scavenging ability range, and the high value measurement limit was such that the measured value was a recovery rate of 95 to 105% of the true value. The concentration was within the range.

(Preparation of reagent)
A second reagent of a neutral fat measuring reagent having the following composition was prepared.
Second reagent PIPES-NaOH 50 mM pH 7.0
Magnesium chloride hexahydrate 0.2g / L
Calcium chloride 0.1g / L
ADPS 0.3g / L
Peroxidase (PEO-301 manufactured by Toyobo) 2.9 U / mL
Lipase (Toyobo LPL-314) 2U / mL

(Measurement method)
A Hitachi 7170 automatic analyzer was used. 180 μL of the first reagent was added to 2.1 μL of the sample, and incubated at 37 ° C. for 5 minutes to obtain a first reaction. Thereafter, 90 μL of the second reagent was added and incubated for 5 minutes to obtain a second reaction. The absorbance at 600 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid amount and the difference between the absorbances was obtained.
The results were calculated from the measured absorbance of purified water and a 200 mg / dL aqueous solution of triolein and determined as the neutral fat concentration.

Results shown in Table 3. In the comparative example, the glycerol scavenging ability was reduced by 40% and the high value measurement limit was decreased by 35%, whereas in the examples, the glycerol scavenging ability was substantially not decreased at 90% or more in both the high value measurement limit.

(Example 4)
For the first reagent solution of the magnesium measurement reagent of Example 2, the following reagents were combined as the second reagent, and a 5 mg / dL aqueous solution of magnesium was measured with each reagent by the following measurement method, and the measurement sensitivity was calculated. The sensitivity after storage for one week before storage was shown as a relative value (%).

(Preparation of reagent)
First magnesium measurement reagents having the following compositions were prepared.
First reagent PIPES-NaOH 50 mM pH 6.8
Disodium ethylenediaminetetraacetate 3g / L
Adenosine triphosphate 2Na salt 1.5 g / L
Triton X-100 1g / L
TODB 0.2g / L
Flavin adenine dinucleotide 2Na salt 5 μmol / L
Glycerophosphate oxidase (G3O-311 manufactured by Toyobo) 10 U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo) 1 U / mL
Peroxidase (PEO-301 manufactured by Toyobo) 3U / mL

(Measurement method)
A Hitachi 7170 automatic analyzer was used. 180 μL of the first reagent was added to 5.8 μL of the sample, and incubated at 37 ° C. for 5 minutes to obtain a first reaction. Thereafter, 90 μL of the second reagent was added and incubated for 5 minutes to obtain a second reaction. The absorbance at 600 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid amount and the difference between the absorbances was obtained.
The results were calculated from the measured absorbance of purified water and a 5 mg / dL aqueous magnesium solution, and determined as the cholesterol concentration.

Results shown in Table 4. In the comparative example, the sensitivity was decreased by 32%, whereas in the example, almost no decrease was observed.

(Example 5)
Immediately after preparation, control serum values (L concealer N and L concealer AN) after storage at 10 ° C. for 12 months were measured by combining the first reagent solution and the second reagent of the neutral fat measurement reagent of Example 3, and after storage. Was determined as a relative value (%) to the serum value immediately after preparation.

Results—shown in Table 5. In the comparative example, the sensitivity was lost and the calculation of the serum value was impossible. In the Examples, 103% for L-Concealer N and 99.3% for L-Concealer AN showed almost no change in serum values before and after storage.

(Example 6)
N-methylisothiazolone (manufactured by Roche), glycerol kinase (derived from Thermus Flavas, GYK-311 manufactured by Toyobo Co., Ltd., derived from Streptomyces canus Genzyme, derived from Geotricum candy dam Roche, derived from Bacillus stearothermophilus Neutral fat measurement reagent of Example 1 to which Roche) was added The first test solution was stored at 9 ° C. and 35 ° C. for 2 weeks, and the residual activity (the ratio of the activity value after storage to the activity value immediately after dissolution) was examined. . Two cases were examined, one in which sucrose was added to the reagent at 5 g / L and the other was not, and compared with the case where no preservative was added.

(Preparation of reagent)
A first reagent of a neutral fat measuring reagent having the following composition was prepared.
First reagent PIPES-NaOH 50 mM pH 6.6
MgCl2 0.2g / L
Adenosine triphosphate 2Na salt 0.9 g / L
Emulgen A60 2g / L
Triton X-100 1g / L
4-aminoantipyrine 0.1 g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Glycerol kinase 3U / mL
Glycerophosphate oxidase (G3O-311 manufactured by Toyobo) 5 U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo) 3 U / mL
Catalase (Toyobo) 200U / mL
N-methylisothiazolone 100mg / L

Results—shown in Tables 6 and 7. When preservatives were added, the enzyme from Geotricum candy dam had already lost its enzymatic activity immediately after preparation. Upon storage at 9 ° C., the enzyme from Streptomyces canus was relatively stable in this. On the other hand, when stored at 35 ° C., the enzymes derived from Thermus flavus and the enzymes derived from Bacillus stearothermophilus were relatively stable. When sucrose was added, the action of further stabilizing the enzyme derived from Thermus flavus and the enzyme derived from Bacillus stearothermophilus at 35 ° C was observed.

The biological component measurement method and the reagent of the present invention have high resistance to preservatives, and have characteristics such as substantially no turbidity during storage and no deterioration in various properties. It is excellent as a diagnostic agent used in the field, and greatly contributes to the industry.

Claims (7)

中 A method for measuring neutral fat, characterized in that the effect of preservative performance and suppression of reagent performance decrease are compatible at the same time during storage in the presence of a preservative. 2. The method according to claim 1, wherein the suppression of the deterioration of the reagent performance is at least one selected from the following: (1) substantially no turbidity; (2) substantially no increase in the reagent blank; (4) Substantially no lowering of the high value measurement limit (5) Substantially no lowering of the enzyme activity contained in the reagent マ グ ネ シ ウ ム A method for measuring magnesium, characterized in that the effect of preservative performance and the suppression of reagent performance deterioration are compatible at the same time during storage in the presence of a preservative. 4. The method according to claim 3, wherein the suppression of the decrease in reagent performance is at least one selected from the following: (1) substantially no increase in the reagent blank; (2) substantially no decrease in sensitivity; The enzyme activity contained in the reagent does not decrease The method according to any one of claims 1 to 4, wherein the preservative is a preservative that acts directly on a protein. 中 A neutral fat measurement reagent characterized in that the antiseptic performance effect and the suppression of reagent performance degradation are compatible at the same time during storage in the presence of a preservative. マ グ ネ シ ウ ム A reagent for measuring magnesium, characterized in that the effect of antiseptic performance and suppression of reagent performance degradation are compatible at the same time during storage in the presence of preservatives.