JP4035786B2 - Biological component measuring method having antiseptic resistance and its reagent - Google Patents

Description

  The present invention relates to a method for measuring neutral fat or magnesium and its reagent. In particular, the present invention relates to a reagent for measuring neutral fat or magnesium in biological components used for clinical diagnosis.

  As a diagnostic agent used in the clinical laboratory field, there is a biochemical measurement reagent using an enzyme reaction. In this reagent, since the enzyme is unstable in the solution, the enzyme and the unstable substance other than the enzyme were lyophilized and dissolved in a solution before use. For this reason, there is a problem that cost and manufacturing time are required because freeze-drying is performed on the manufacturing side, and that dissolution is required on the user side. On the other hand, in recent years, an unprepared non-prepared liquid reagent has been developed in the field of biochemical examination, and the problems associated with freeze-drying have been solved. This liquid reagent has been completed as a reagent that can be stored for a long time in a solution state by stabilizing raw material enzymes, coenzymes, etc. by various methods, and is now mainstream in the domestic biochemical measurement reagent market. Yes.

In the development of non-prepared liquid reagents, one of the development points that should be newly added from the conventional freeze-dried reagents is to improve the antiseptic ability at the time of distribution. In order to improve the antiseptic ability, various additives have been studied and results have been reported (for example, see Patent Document 1). However, in selecting a preservative, while taking into consideration antiseptic performance such as antibacterial activity and long-term stability of the preservative itself, on the other hand, the influence on the stability of the raw enzyme and coenzyme cannot be ignored, In fact, it has been found that the preservative may degrade the component enzymes and the like due to the compatibility of the preservative and the enzyme, and may affect the reagent performance such as precision and accuracy. Thus, it is a very difficult task to achieve both sufficient antiseptic performance and reagent stability.
JP 7-78005

Examples of the reagent whose performance of the measuring reagent deteriorates due to the preservative include a neutral fat measuring reagent and a magnesium measuring reagent (see, for example, Patent Documents 2 and 3). Neutral fat is generally measured by measuring glycerophosphate or ADP produced by allowing lipase and glycerol kinase to act on neutral fat. Among them, a method is widely used in which hydrogen peroxide generated by allowing glycerophosphate oxidase to act on glycerophosphate acts on a chromogen under peroxidase to produce a quinone dye. In addition, when measuring in a two-reagent system using an automatic analyzer or the like, ascorbic acid oxidase is added to the first reagent and ascorbic acid, which is a substance that interferes with the measurement, is erased in this field. It is. In addition, catalase is often added to eliminate endogenous glycerol in the art.
The reagent for measuring magnesium is generally measured by measuring glycerophosphate or ADP which produces glycerol kinase activity activated by magnesium.
Shoko 58-5679 JP-A 64-30597

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

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

In order to achieve the above object, the present inventors have studied the interaction between components such as enzymes and dyes in the reagent and the preservative, and as a result of intensive studies, have selected an appropriate combination of enzyme and preservative. As a result, the component enzyme in the reagent deteriorates due to preservatives, causing turbidity during storage in the reagent for measuring neutral fat, increasing the reagent blank during the reaction, and reducing the ability to eliminate endogenous free glycerol. When the high-value measurement limit is lowered, turbidity occurs during storage in the magnesium measurement reagent, the reagent blank rises during the reaction, the sensitivity is lowered, and the high-value measurement limit is lowered to reduce the precision of the measurement reagent. As a result, the present invention was completed. That is, the present invention has the following configuration.
(1) Neutral fat measurement method characterized by having high resistance to preservatives and substantially no 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 substantially no reduction in glycerol scavenging ability during storage Neutral fat measurement method characterized by the following: (4) Neutral fat measurement method characterized by having high resistance to preservatives and that the high-value measurement limit does not substantially decrease during storage (5) Preservation Magnesium measuring method characterized by having high resistance to an agent and substantially no turbidity during storage (6) High resistance to an antiseptic and substantially during storage Magnesium measurement characterized by no rise in reagent blank Method (7) Magnesium measurement method characterized by having high resistance to preservatives and substantially no decrease in sensitivity during storage (8) High resistance to preservatives and storage Neutral fat measurement reagent (9) characterized by substantially no turbidity in the interior (9) It has high resistance to preservatives, and there is substantially no increase in reagent blank during storage The neutral fat measurement reagent (10) has a high resistance to a preservative and the glycerol scavenging ability is not substantially reduced during storage. And has a high resistance to a preservative for neutral fat, characterized in that the high-value measurement limit does not substantially decrease during storage, and substantially during storage Magnesium measurement characterized by no turbidity Reagent (13) has high resistance to preservatives, and has a high resistance to magnesium measurement reagent (14) preservatives characterized by substantially no increase in reagent blanks during storage, Magnesium measuring reagent characterized in that sensitivity does not substantially decrease during storage

In other words, the present invention has the following configuration.
[1]
Neutral fat measurement method [2] characterized in that both preservative performance effect and suppression of reagent performance decrease are compatible during storage in the presence of a preservative.
Suppression of reagent performance is at least one selected from the following: [1] Method (1) No substantial turbidity (2) No substantial increase in reagent blank (3) Substantially Glycerol scavenging ability is not lowered (4) High value measurement limit is not lowered (5) Enzyme activity contained in the reagent is not substantially lowered [3]
Magnesium measuring method [4], characterized in that both antiseptic performance effects and suppression of reagent performance degradation are compatible during storage in the presence of a preservative.
Inhibition of reagent performance degradation is at least one selected from the following: [3] Method (1) No substantial increase in reagent blank (2) No substantial decrease in sensitivity (3) Substantially The enzyme activity contained in the reagent should not decrease [5]
The method [6] according to any one of [1] to [4], wherein the preservative is a preservative that directly acts on the protein.
Neutral fat measurement reagent [7], characterized in that both antiseptic performance effects and suppression of reagent performance degradation are compatible during storage in the presence of a preservative.
Magnesium measuring reagent, characterized in that both antiseptic performance effects and suppression of reagent performance degradation are compatible during storage in the presence of preservatives

  According to the present invention, it is possible to provide a neutral fat measurement reagent and a magnesium measurement reagent that have high resistance to preservatives and maintain the precision and accuracy of the measurement 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, It is a method for measuring neutral fat that has high resistance to water and does not generate turbidity during storage.

  When antibiotics are used as preservatives, microorganisms can relatively easily suppress the membrane permeability of the substance or acquire the ability to decompose and detoxify the substance, thus preventing the generation of resistant bacteria. It is often considered that the antiseptic effect of the reagent is often lost. On the other hand, a preservative capable of directly acting on a protein is more preferable in that it is considered difficult for a microorganism to obtain resistance, and is likely to be widely used in the future. However, such a preservative that directly acts on the protein naturally acts on the coexisting enzyme protein, and thus may be destabilized depending on the structure of the protein. Thus, resistance to a preservative 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 isothiazoline such as 2-methyl-4-isothiazolin-3-one-hydrochloride (also referred to as N-methylisothiazolone, abbreviation MIT). Compounds, 2-hydroxypyridine-N-oxide (abbreviation HPO), chloroacetamide (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 a magnesium measurement reagent, requires many elements such as enzymes and pigments, but the effects of preservatives on each element are different. This is to reduce the number of affected elements as much as possible.

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

  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 the like. These are Procrine 150 (manufactured by Superco), Procrine 300 (manufactured by Superco), Actiside MBS (manufactured by So Japan), Actiside B20 (N) (manufactured by So Japan), MIT (Manufactured by Roche) Sigma, etc.) are generally commercially available. In addition, the aspect which does not add antiseptic | preservatives other than an isothiazoline type compound in a reagent for the reason already stated is still more preferable. In the method for measuring neutral fat of the present invention, the isothiazoline-based compound is excellent both in terms of antiseptic ability and influence on the stability of the reagent.

  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. The concentration of the preservative added at this time is not particularly limited, but is desirably a concentration that can provide a sufficient antiseptic performance effect. Naturally, the addition concentration of the preservative varies depending on the kind of the preservative and the composition of the reagent to be added, and the appropriate addition concentration can be appropriately determined by those skilled in the art. The concentration range is not particularly limited as long as it has sufficient antiseptic properties and can be used for diagnostic drug applications. For example, the lower limit concentration in a 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%.

  A sufficient antiseptic performance effect can be seen, for example, by the absence of bacterial growth by the following antibacterial test. In the antibacterial test method, a measurement reagent to which an antiseptic is added is used as a test reagent, which 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 fluid is a standard strain such as Pseudomonas sp. Prepare a suspension so that the absorbance at 660 nm is 0.5 Abs. After inoculating 50 μL of this bacterial solution with respect to 4 mL of the test reagent, shake at 25 or 37 ° C. for 3 days. The determination is that the absorbance at 660 nm of the test reagent is compared before and after storage, and if there is a significant increase in absorbance, it is determined that there is no antiseptic effect. If there is no significant difference in absorbance variation, it is determined that there is no growth of bacteria. Judged as effective.

  The high resistance to the preservative means that the antiseptic performance effect and the suppression of the decrease in reagent performance are compatible in the presence of the preservative during the storage. For example, a preservative whose resistance is to be tested is added to a certain composition, and during storage, degradation of reagent performance such as accuracy, precision, and measurement range is substantially suppressed, and substantially suspended matter and sediment 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 storage period can be arbitrarily set within the scope 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 implemented, it can be replaced with an accelerated test at 35 to 37 ° C. for one week or longer. 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, neutral fat measurement reagents become turbid during storage, reagent blanks rise during the reaction, endogenous free glycerol scavenging ability decreases, and high value measurement A phenomenon such as a decrease in the limit is seen. In the magnesium measurement reagent, turbidity occurs during storage, reagent blank rises during the reaction, sensitivity decreases, and the high value measurement limit decreases. It can be evaluated by observing the transition of these phenomena whether or not the reagent performance deterioration is suppressed among the resistance to the preservative.

  These phenomena are thought to be caused by deterioration of various elements such as enzymes and pigments. The decrease in endogenous free glycerol and the decrease in the high measurement limit are mainly due to the slowdown of at least one of the multi-stage enzymatic reactions due to enzyme inactivation, thereby delaying the follow-up of the reaction. Responsible. In the neutral fat measurement reagent, examples of the enzyme involved in glycerol elimination include glycerol kinase, glycerophosphate oxidase, and catalase. Examples of the enzyme involved in the measurement include lipase, glycerol kinase, glycerophosphate oxidase, and peroxidase. In addition, turbidity during storage and reagent blank increase during the reaction are mainly caused by enzyme protein being denatured and insolubilized by inactivation, but in the case of turbidity, other substances such as surfactants and salts are further added. In some cases, the pigments may be insolubilized by the action of the preservative, and in the case of increasing the blank, the dyes may be further self-condensed by the action of the preservative.

  Conventionally, when an isothiazoline-based compound is used as a preservative, it is considered that glycerol kinase is mainly brought about by deterioration due to the preservative. Therefore, for example, when an isothiazoline-based compound is used as a preservative in the present invention, whether the reagent performance deterioration is suppressed among the resistance to the preservative is determined according to the concentration of glycerol kinase having a concentration of 0.1 to 20 U / mL. It can also be evaluated by looking at the deterioration due to. Whether or not the reagent performance deterioration is suppressed is determined by whether or not “the enzyme activity contained in the reagent is not substantially reduced”. When the preservative is 100 mg / L at 25 ° C. for 1 week, the residual ratio of glycerol kinase activity 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 enzyme activity before and after storage under the above conditions may be compared after adding the preservative of the above concentration to the reagent composition to be evaluated.

  As described above, since the effect on the enzyme varies depending on the type of the preservative used, whether or not the reagent performance deterioration is suppressed among the resistance to the preservative is appropriately determined by the enzyme affected by the preservative. It can be selected and evaluated in the same manner as glycerol kinase. The enzyme serving as an index 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 reagent performance deterioration is suppressed is determined by the enzyme activity remaining rate of 70% or more, preferably 80% or more, more preferably 90% or more when coexisting at 25 ° C. for 1 week at a concentration of 100 mg / L of the preservative. It is. The same applies to the magnesium measurement reagent.

  In the present invention, “substantially no turbidity” means that turbidity does not occur at all or hardly turbidity occurs and is kept below a certain level. The “certain level” is appropriately determined based on common knowledge of those skilled in the art. As a method for evaluating turbidity during storage, it can be evaluated by visual observation, absorbance measurement at 660 nm, measurement by a particle counter (manufactured by Kurabo Industries), or the like. Among these, a particle counter is preferable from the viewpoint of minimum detection sensitivity. The total count of 5 to 150 μm at 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 conventionally used test method such as an evaluation method used in the field of quantitative inspection or analytical chemistry can be appropriately employed.

  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 triglycerides, which has a high resistance to the absence of reagent blanks during storage.

  In the present invention, the reagent blank refers to the absorbance measured by the measurement wavelength at the time when the measurement is completed when a sample not containing purified water or a measurement target is measured. “Substantially no increase in the reagent blank” means that there is no or almost no increase in the blank, and is kept below a certain level. The increase after storage of the reagent blank in the present invention 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 conventionally used test method such as an evaluation method used in the field of quantitative inspection or analytical chemistry can be appropriately employed.

  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, It is a method for measuring neutral fat that has a high resistance to glycerol and does not reduce the ability to eliminate glycerol during storage.

  In the present invention, the glycerol scavenging ability is the ability to convert (eliminate) free glycerol, which becomes a disturbing factor when included in a sample in the measurement of triglyceride, to a substance not involved in the reaction before the measurement of triglyceride. I mean. “Substantially no reduction in glycerol-eliminating ability” means that the erasing ability is not lowered or hardly lowered and is suppressed to a certain level or more. The “certain level” is appropriately determined according to common knowledge of those skilled in the art. Usually, 100 mg / dL in terms of triolein is sufficient, but it is preferably 2000 mg / dL or more, more preferably 3500 mg / dL or more. Under these conditions, the decrease in glycerol scavenging ability in the present invention after storage with respect 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) Dilute glycerol (manufactured by Nacalai Tesque) with purified water so that the concentration is evenly spaced to produce a dilution series of 10 levels or more.
(2) The above sample is measured twice or more using a neutral fat measurement reagent, and the average value is calculated. A Hitachi 7170 automatic analyzer is used for the measurement. Add 180 μL of the first reagent to 2.1 μL of the sample, and incubate at 37 ° C. for 5 minutes for the first reaction. Thereafter, 90 μL of the second reagent is added and incubated for 5 minutes to form the second reaction. The absorbance at 600 nm is measured by the two-point end method by taking the difference in absorbance obtained by correcting the absorbance of the first reaction and the second reaction. The absorbance measurement result is calculated from the measured absorbance of purified water and 200 mg / dL triolein aqueous solution, and obtained as the neutral fat concentration.
(3) The measured value should be zero if it is completely erased. Plot the measured values of triolein at each level to see how far the zero is maintained. When the measured value exceeds 10 mg / dL (5% of 200 mg / dL triolein), it is determined that there is no glycerol scavenging ability at concentrations higher than this concentration level, and the concentration below one level is defined as glycerol scavenging ability. To 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 triglycerides, which has a high resistance to odor and does not lower the high value measurement limit.

In the present invention, the high value measurement limit refers to the maximum value of the concentration in 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.
“Substantially the high value measurement limit does not decrease” means that the measurement limit does not decrease or hardly decreases and is suppressed to a certain level or more. The “certain level” is appropriately determined according to common knowledge of those skilled in the art, but in general, it may be a 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, more preferably 2500 mg / dL or more. Under these conditions, the decrease after storage of the high measurement limit in the present invention 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) is diluted with purified water so that the concentration is evenly spaced to prepare a dilution series of 10 levels or more.
(2) The above sample is measured twice or more using a neutral fat measurement reagent, and the average value is calculated. A Hitachi 7170 automatic analyzer is used for the measurement. Add 180 μL of the first reagent to 2.1 μL of the sample, and incubate at 37 ° C. for 5 minutes for the first reaction. Thereafter, 90 μL of the second reagent is added and incubated for 5 minutes to form the second reaction. The absorbance at 600 nm is measured by the two-point end method by taking the difference in absorbance obtained by correcting the absorbance of the first reaction and the second reaction. The absorbance measurement result is calculated from the measured absorbance of purified water and 200 mg / dL triolein aqueous solution, and obtained as the neutral fat concentration.
(3) Plot the value obtained by dividing the measured value of triolein at each level by the measured value at the first level to see how far the proportional relationship is maintained. When the value deviates from ± 5% of the theoretical value, it is determined that there is no linearity at higher concentrations including this concentration level, and the concentration below one level is set as the high value measurement limit.

  Further, 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, glycerol or the like as a second reagent, which is highly resistant to preservatives. It is a magnesium measuring reagent that is resistant and does not generate turbidity during storage.

  Further, 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, glycerol or the like as a second reagent, which is highly resistant to preservatives. It is a magnesium measuring reagent that is resistant and does not rise in reagent blanks during storage.

  The increase after storage of the reagent blank in the present invention before storage is 0.1 Abs or less, preferably 0.05 Abs or less, more preferably 0.01 Abs or less.

  Further, 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, glycerol or the like as a second reagent, which is highly resistant to preservatives. It is a magnesium measuring reagent that is resistant and does not lose sensitivity during storage.

In the present invention, the sensitivity refers to an absorbance obtained by subtracting the reagent blank absorbance from the absorbance when a sample having a known concentration in a range where the quantitativeness is guaranteed.
“Substantially no decrease in sensitivity” means that the sensitivity is not decreased or hardly decreased, and is suppressed to a certain level or more. The “certain level” is appropriately determined according to common knowledge of those skilled in the art, but in general, it may be a level that satisfies the reagent specifications. Under these conditions, the decrease in sensitivity after storage relative to the storage before storage in the present invention is 30% or less, preferably 20% or less, more preferably 10% or less.

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

  Further, 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, glycerol or the like as a second reagent, which is highly resistant to preservatives. This is a magnesium measuring method that is resistant and satisfies at least two of the following [1] to [3]. [1] Substantially no turbidity occurs during storage, [2] No substantial increase in reagent blank during storage, [3] No substantial decrease in sensitivity during storage.

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

  The origin of such an enzyme is not particularly limited. Examples of animal origins include human, rat liver, and pigeon liver. In addition, if the microorganism is a yeast or a microorganism, the genus Saccharomyces (for example, Saccharomyces cerevisiae), Candida (for example, Candida mycoderma), or Bacillus (for example, Bacillus subtilis (Bacillis) ), Bacillus stearothermophilus, Thermus (e.g., Thermus flavus), Cellulomonas (e.g., Cellulomonas flavigena, Cellulomones sp. Escherichia (eg, esche Escherichia coli, Arthrobacter spp. (Eg Arthrobacter sp.), Flavobacterium spp. (Eg Flavobacterium meningosepticum), Streptomy Examples include the genus Seth (for example, Streptomyces canus) and Geotricum candidum. Any of these may be commercially available.

  Of these, the Cellulomonas genus, Flavobacterium genus (Flavobacteria membranum) are preferably used in terms of maintaining the stability of the reagent even when coexisting with antiseptics, particularly those that act directly on proteins. Ningocepticum), Arthrobacter sp. (Arthrobacter sp.), Thermus sp., Bacillus sp. (Bacillus stearothermophilus). More preferred are those of the genus Cellulomonas, Thermus, and Bacillus (Bacillus stearothermophilus). Most preferably, those extracted from Cellulomonas sp. Are used. The above-mentioned enzymes include those produced from genetically modified microorganisms in which these genes are incorporated into other microorganisms.

One of the reasons for the above difference is considered to be due to the number and position of a portion susceptible to reaction, for example, a sulfur-containing amino acid residue such as cysteine or methionine in an 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, in sarcosine oxidase, there is an example in which stabilization of various substances is achieved by substituting cysteine residues with other amino acids (see, for example, Patent Document 5). Therefore, when there are sulfur-containing amino acids, especially when sulfur-containing amino acids are present on the enzyme surface, especially when sulfur-containing amino acids are present near the active center, coenzyme, or substrate binding site, the effects of preservatives and other chemicals Suggested to be more susceptible. As for the number of sulfur-containing amino acids, unless the other conditions are significantly different, in principle, it is considered that the smaller the number of sulfur-containing amino acids, the more preservative resistant.
Japanese Patent Laid-Open No. 11-9279 Japanese Patent No. 3132618

  The lower limit of the concentration of 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.

  Examples of the buffer used in the present invention include Tris buffer, phosphate buffer, and GOOD buffer. Of these, Tris buffer and phosphate buffer have the advantage of being inexpensive, although the pH is likely to vary depending on the concentration and temperature. On the other hand, examples of the GOOD buffer solution having a small pH fluctuation include MES, Bis-Tris, ACES, BES, MOPS, PIPES, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS. Any of these may be commercially available.

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

  A surfactant, saccharide, chelating agent, coenzyme and the like may be further added to the measuring reagent of the present invention. Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and zwitterionic surfactants. Examples of the saccharide include mannitol, sorbitol, cyclodextrin and derivatives thereof. Examples of the chelating agent include ethylenediaminetetraacetic acid and its salt. Examples of coenzymes include flavin adenine dinucleotide. Any of these may be commercially available. The purpose of these additions is not particularly limited, but is preferably a kind and concentration having an effect of stabilizing enzymes and reagents. Moreover, it is preferable that the additive itself is a kind and density | concentration which do not receive deterioration by a preservative.

  The measurement reagent of the present invention may contain other reagents necessary for measurement. Examples of the neutral fat measurement reagent may include lipase, glycerol kinase, glycerophosphate oxidase, ATP, magnesium, peroxidase, and a color source. Further, for example, lipase, glycerol kinase, ATP, ADP-dependent hexokinase, glucose-6-phosphate dehydrogenase, oxidized nicotinamide adenine dinucleotide can be contained. Further, as the magnesium measuring reagent, generally, glycerol kinase, glycerophosphate oxidase, ATP, chelating agent, peroxidase, and color source can be contained.

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

  Furthermore, in this invention, you may add saccharides. 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 monocaprate, sucrose monocholate and the like. Among them, compounds having a sucrose skeleton, such as sucrose, lactose sucrose, raffinose, inulooligosaccharides, sucrose monocaprate, sucrose monocholate and the like are preferably used. Of these, sucrose is preferred. In any case, commercially available products can be used. Although there is no restriction | limiting in particular in the addition density | concentration of saccharides, Preferably, it is 0.01 times amount (W / W) or more with respect to antiseptic | preservative, and a preferable minimum is 0.05 times amount (W / W), Preferably The upper limit is 500 times the amount (W / W). In particular, when an isothiazoline compound is used as a preservative, resistance (stabilizing effect) is increased, which is preferable.

The activity of glycerol kinase is preferably measured under the following measurement conditions.
Using glycerol as a substrate, enzyme activity was measured by the amount of glycerol-3-phosphate produced. 0.5% 4-aminoantipyrine aqueous solution 0.2 ml, 1.5% phenol aqueous solution 0.2 ml, glycerol-3-phosphate oxidase 200 U, peroxidase 80 U, ATP 48.4 mg, 0.1 M HEPES buffer (pH 7.9) Was added to make a total volume of 21 ml, which was used as a stock solution for the following measurement. For each reaction, 3 ml of this measurement stock solution was taken, 50 μl of 0.3 M glycerol aqueous solution and 100 μl of 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, an enzyme dilution solution (20 mM potassium phosphate buffer solution containing 0.2% bovine serum albumin, pH 7.5) was added in an amount of 100 μl instead of the enzyme solution, and the same operation as described above was performed to determine the amount of change in absorbance per minute. It was determined (ΔODblank).
The enzyme activity of glycerol kinase was calculated from the obtained change in absorbance based on the following formula. The amount of enzyme that phosphorylates 1 micromole of glycerol per minute under the above conditions is defined as 1 unit (1 U).
Formula activity value (U / ml) = {ΔOD / min (ΔODtest−ΔODblank) × 3.15 (ml) × dilution factor} / {13.3 × 1/2 × 1.0 (cm) × 0.1 (Ml)}
3.15 ml = reaction mixture volume 13.3 = molar extinction coefficient of quinone dye under the above measurement conditions 1/2 = 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 solution amount

Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not specifically limited by an Example.
Example 1
The following neutral fat measuring reagent containing N-methylisothiazolone (Roche) and glycerol kinase (Cellulomonas SP-derived Toyobo Co., Ltd., GYK-301) was stored at 25 ° C. for 1 week, and the remaining activity ( The ratio of the activity value after storage to the activity value immediately after dissolution) was examined. In the comparative example, glycerol kinase was replaced by Cellulomonas sp and derived from Thermus fullerbus (Toyobo Co., Ltd.).

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

Results are shown in Table 1. In the comparative example, the activity decreased to about 40%, whereas in the example, good stability of 90% or more was exhibited after storage.

(Example 2)
The following magnesium measuring reagent to which N-methylisothiazolone (Roche) and glycerol kinase (GYK-301 from Cellulomonas sp.) Were added was stored at 25 ° C. for 1 week, and the remaining activity (immediately after dissolution) The ratio of the activity value after storage to the activity value) was examined. In the comparative example, glycerol kinase was replaced by Cellulomonas sp and derived from Thermus fullerbus (Toyobo Co., Ltd.).

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

Results are shown in Table 2. In the comparative example, the activity decreased to about 60%, whereas in the example, good stability of 90% or more was exhibited after storage.

(Example 3)
For the first reagent of the neutral fat measurement reagent of Example 1, the following reagents were combined as the second reagent, and the following measurement method was used to measure each of the reagents at a glycerol 5000 mg / dL (triolein conversion value) level of 10; Further, 10 levels of dilution of 3000 mg / dL of neutral fat were measured, glycerol elimination ability and high limit of measurement were calculated, and the performance after storage relative to 25 ° C. for 1 week before storage was shown as a relative value (%). The determination of glycerol scavenging ability is determined by determining the maximum concentration of the glycerol dilution level that is 3 mg / dL or less as a measured value as the scavenging ability range, and the high value measurement limit is a recovery rate of 95 to 105% of the measured value with respect to the true value. The concentration was within the range.

(Preparation of reagents)
A second reagent for measuring neutral fat 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 (Toyobo PEO-301) 2.9 U / mL
Lipase (Toyobo LPL-314) 2U / mL

(Measurement method)
A Hitachi 7170 automatic analyzer was used. The first reaction was carried out by adding 180 μL of the first reagent to 2.1 μL of the sample and incubating at 37 ° C. for 5 minutes. Thereafter, 90 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. 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 volume and the difference between the absorbances was taken.
The result was calculated from the measured absorbance of purified water and 200 mg / dL triolein aqueous solution, and obtained as the neutral fat concentration.

Results are shown in Table 3. In the comparative example, the glycerol scavenging ability was reduced by 40% and the high value measurement limit was reduced by 35%, whereas in the examples, the glycerol scavenging ability was 90% or more at the high value measurement limit and almost no reduction was observed.

Example 4
With respect to the first reagent solution of the magnesium measuring reagent of Example 2, the following reagents were combined as the second reagent, a magnesium 5 mg / dL aqueous solution was measured with each reagent by the following measuring method, the measurement sensitivity was calculated, and 25 ° C. The sensitivity after storage relative to the storage before 1 week is shown as a relative value (%).

(Preparation of reagents)
First reagents for magnesium measurement reagents having the following compositions were prepared.
First reagent PIPES-NaOH 50 mM pH 6.8
Ethylenediaminetetraacetic acid disodium 3g / L
Adenosine triphosphate 2Na salt 1.5g / 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 Co., Ltd.) 10 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Peroxidase (Toyobo PEO-301) 3 U / 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 prepare the first reaction. Thereafter, 90 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. 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 volume and the difference between the absorbances was taken.
The result was calculated from the measured absorbance of purified water and a 5 mg / dL magnesium aqueous solution, and obtained as a cholesterol concentration.

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

(Example 5)
Control serum values (L Conselaer N and L Conselaer AN) immediately after preparation and after storage for 12 months were measured by combining the first reagent and the second reagent for the neutral fat measurement reagent of Example 3 and after storage. Was obtained as a relative value (%) to the serum value immediately after preparation.

Results are shown in Table 5. In the comparative example, the sensitivity was lost and the serum value could not be calculated. In the examples, L Consera N was 103%, L Consera AN was 99.3%, and there was almost no change in serum values before and after storage.

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

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

Results are shown in Tables 6 and 7. When a preservative was added, the enzyme derived from Geotricham candy dam had already lost its enzyme activity immediately after preparation. When stored at 9 ° C., the enzyme derived from Streptomyces canus was relatively stable. On the other hand, when stored at 35 ° C., the enzyme derived from Thermus flavus and the enzyme derived from Bacillus stearothermophilus were relatively stable. When sucrose was added, a function 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 reagent of the present invention have high resistance to preservatives and have characteristics such as no substantial turbidity during storage, such as no deterioration in performance, and clinical tests. It is excellent as a diagnostic agent used in the field, and greatly contributes to the industry.

Claims (2)

In the presence of N-methylisothiazolone, a magnesium measurement method that uses a magnesium measurement reagent that is compatible with both antiseptic performance effects and suppression of reagent performance degradation continuously during storage,
The magnesium measurement reagent consists of a two-reagent system in which the second reagent contains N-methylisothiazolone, Cellulomonas-derived glycerol kinase, and
The method for measuring magnesium, wherein the suppression of reagent performance deterioration is at least one selected from the following.
(1) The decrease in sensitivity is 30% or less.
(2) Glycerol kinase activity when glycerol kinase having a concentration of 0.1 to 20 U / mL was allowed to coexist at a concentration of 100 mg / L of N-methylisothiazolone at 25 ° C. for 1 week. Residual rate is 70% or more.   In the presence of N-methylisothiazolone, it is a magnesium measuring reagent that has both antiseptic performance effects and suppression of reagent performance deterioration continuously during storage, and the second reagent is derived from N-methylisothiazolone, Cellulomonas A reagent for measuring magnesium comprising a two-reagent system containing glycerol kinase.