JP2004105024A - Method for stabilizing nucleoside or nucleotide or derivative thereof in solution and composition therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stabilizing a nucleoside or a nucleotide or a derivative thereof used for an enzymatic assaying reagent, etc., for a physiologically active substance, e.g. aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, pyruvic acid, a neutral fat, urea nitrogen or an inorganic phosphorus in a biocomponent used for clinical diagnosis in a solution and to provide a composition therefor. <P>SOLUTION: The composition is obtained by making an antibiotic coexist in the solution containing the nucleoside or nucleotide or the derivative thereof and a substance degrading the nucleoside or nucleotide or the derivative thereof as an impurity. Thereby, the nucleoside or nucleotide or the derivative thereof can be kept stable in the solution state over a long period and good reactivity can be maintained for various kinds of samples. <P>COPYRIGHT: (C)2004,JPO

Description

【００３２】
結果　　表１に示す。実施例ではいずれも比較例に対しＡＴＰ残存率が高く良好な安定性を示し、また保存後の中性脂肪の測定値回収率も良好であり反応性を維持した。
【００３３】
（実施例２）
ＡＴＰ・２Ｎａ（ロッシュ社製）１ｍＭを含有する実施例１に記載の中性脂肪測定試薬　第一試液にカナマイシン０．００５％〜０．２％を添加溶解し３５℃で１４日間保存し、ＡＴＰ残存量（溶解直後の濃度に対する保存後の濃度の割合）を検討した。
【００３４】
【表２】

【００３５】
結果　表２に示す。実施例ではいずれも比較例に対しＡＴＰ残存率が高く良好な安定性を示し、また保存後の中性脂肪の測定値回収率も良好であり反応性を維持した。低濃度のカナマイシンでも効果を確認できた。
【００３６】
（実施例３）
イノシン（コウジン社製）１２ｍＭを含む下記の無機リン測定試薬　第ニ試液に各種抗生物質　各０．０５％を添加溶解し３５℃で１４日間保存し、ＡＴＰ残存量（溶解直後の濃度に対する保存後の濃度の割合）を検討した。また、３５℃、１４日間保存した試薬を用いて無機リン濃度４０ｍｇ／ｄＬの試料を測定し、各測定値の理論値に対する回収率を算出した。
【００３７】
（試薬の調製）
下記組成からなる無機リン測定試薬をそれぞれ調製した。
第一試薬
ＰＩＰＥＳ−ＮａＯＨ　　５０ｍＭ　ｐＨ７．０
ＥＤＴＡ・２Ｎａ　０．３ｍＭ
トリトンＸ−１００　　２ｇ／Ｌ
ＡＤＰＳ　　０．１ｇ／Ｌ
キサンチンオキシダーゼ（東洋紡社製ＸＴＯ−２１１）　５Ｕ／ｍＬ
ペルオキシダーゼ（東洋紡社製ＰＥＯ−３０１）　１Ｕ／ｍＬ
アスコルビン酸オキシダーゼ（東洋紡社製ＡＳＯ−３１１）　３Ｕ／ｍＬ
第二試薬
ＰＩＰＥＳ−ＮａＯＨ　５０ｍＭ　ｐＨ７．０
ＥＤＴＡ・２Ｎａ　０．３ｍＭ
トリトンＸ−１００　　２ｇ／Ｌ
４−アミノアンチピリン　　０．３ｇ／Ｌ
イノシン　１２ｍＭ
キサンチンオキシダーゼ（東洋紡社製ＸＴＯ−２１１）　１Ｕ／ｍＬ
ペルオキシダーゼ（東洋紡社製ＰＥＯ−３０１）　１Ｕ／ｍＬ
プリンヌクレオチドホスホリラーゼ（東洋紡社製ＰＮＰ−３０１）　１Ｕ／ｍＬ
【００３８】
（測定法）
日立７１７０形自動分析機を用いた。試料２．１μＬに第一試薬　１８０μＬ添加し３７℃にて５分間インキュベーションし第一反応とした。その後第二試薬を９０μＬ添加し５分間インキュベーションし第二反応とした。第一反応および第二反応の吸光度を液量補正した各吸光度の差をとる２ポイントエンド法で６００ｎｍにおける吸光度を測定した。
結果は、精製水および５ｍｇ／ｄＬ無機リン水溶液の測定吸光度より算出し無機リン濃度として求めた。
【００３９】
【表３】

【００４０】
結果　表３に示す。実施例ではいずれも比較例に対しイノシン残存率が高く良好な安定性を示し、また保存後の無機リンの測定値回収率も良好であり反応性を維持した。
【００４１】
（実施例４）
アデノシン３リン酸（ロッシュ社製）１ｍＭとカナマイシン０．０３、０．０６％とを添加した下記の中性脂肪測定試薬　第一試液に添加溶解し１０℃で１２ヶ月間保存し、ＡＴＰ残存量（溶解直後の濃度に対する保存後の濃度の割合）を検討した。また、１０℃、１２ヶ月間保存した試薬を用いて中性脂肪濃度２０００ｍｇ／ｄＬの試料を測定し、各測定値の理論値に対する回収率を算出した。
【００４２】
【表４】

【００４３】
結果　　表４に示す。実施例ではいずれも比較例に対しＡＴＰ残存率が高く良好な安定性を示し、また保存後の中性脂肪の測定値回収率も良好であり反応性を維持した。長期保存安定性も良好であった。
【００４４】
【発明の効果】
本発明においては、ヌクレオシドまたはヌクレオチドまたはそれらの誘導体を含む溶液に、抗生物質を共存させることにより、溶液中のヌクレオシドまたはヌクレオチドまたはその誘導体を液状状態で長期間安定に保ち、かつ各種試料に対し良好な反応性を維持する方法およびその組成物を提供できる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for stabilizing a nucleoside or a nucleotide or a derivative thereof in a solution and a composition thereof. Especially used for biologically active substances in biological components used for clinical diagnosis, for example, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, pyruvate, neutral fat, urea nitrogen, enzymatic measurement reagent for inorganic phosphorus, etc. And a composition thereof.
[0002]
[Prior art]
As a method for stabilizing a nucleoside or a nucleotide or a derivative thereof in a solution, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) is used in an aqueous carbonate buffer solution having a pH of 9.4 to 10.6. And stabilization by coexisting with glycerin (for example, see Patent Document 1). Further, NADH, oxidized nicotinamide adenine dinucleotide (NAD), NADPH, oxidized nicotinamide adenine dinucleotide phosphate (NADP), adenosine triphosphate (ATP), and adenosine diphosphate (ADP) were added at pH 6 to 8.5 at pH 5 to 8.5. It is disclosed that the composition contains a non-reactive and water-miscible organic solvent such as ethanol at% V / V or more and / or a water-soluble polymer such as gelatin (for example, see Patent Documents 2 to 4). Further, it is disclosed that ATP contains an alkali metal azide or an alkaline earth metal azide (for example, see Patent Document 5). Further, it is disclosed that NADH and NADPH coexist with a salt generated by a reaction with an amine base, a strong base, a monovalent or divalent cation, a weak acid, and a strong base (for example, see Patent Document 6). Further, it is disclosed that NAD (H) and NADP (H) contain a chelating agent and an azide in a buffer solution having a pH of 6.5 to 8 (for example, see Patent Document 7). Further, it is disclosed that a basic aqueous solution of NADH or NADPH contains a borate cis-hydroxyalkyl binding compound such as boric acid in an amount equal to or greater than that of NADH or NADPH (for example, see Patent Document 8). Further, it is disclosed that NADH contains an alkali metal or ammonium carbonate / bicarbonate buffer at pH 9.5 to 11 (for example, see Patent Document 9). However, these methods are limited to short-term stability in solution and have poor long-term stability. Further, it mainly has an effect on the stability of NADH and ATP alone, and the stability of components other than these coenzymes or the reactivity of enzymes or the like is not considered.
[0003]
On the other hand, as a method different from the above-mentioned stabilizer, an enzyme capable of reacting a coenzyme conversion product generated from a destabilized coenzyme such as NADH or NADPH with the product to generate a coenzyme, A method for maintaining a coenzyme content in the presence of a substrate is disclosed (for example, see Patent Documents 10 to 13). However, these methods are effective when degradation products such as NADH or denatured products are identified, but in fact, there are some factors for destabilization and these are not sufficient.
[0004]
On the other hand, as a degradation mode of NADH, there is degradation of NADH to NMNH (reduced nicotinamide mononucleotide) by nucleotide pyrophosphatase or the like existing as a contaminant. Since this reaction has a maximum absorption at 340 nm like NADH, deterioration cannot be confirmed from the absorption characteristics, and NMNH has a problem of inhibiting the enzymatic reaction. On the other hand, it is disclosed that a chelating agent is allowed to coexist under conditions of pH 7.5 to 11 (for example, see Patent Document 14). However, this method is not sufficient when long-term storage is considered, and it is necessary to increase the content of the chelating agent in order to obtain a sufficient effect, which adversely affects enzymes to be measured or enzymes used as reaction components. There is a disadvantage.
[0005]
[Patent Document 1]
JP-B-55-47879 [Patent Document 2]
Japanese Patent Publication No. 58-43078 [Patent Document 3]
JP-A-53-52685 [Patent Document 4]
JP-A-60-105494 [Patent Document 5]
JP-B-54-4280 [Patent Document 6]
JP-A-55-42599 [Patent Document 7]
Japanese Patent Publication No. 2-7319 [Patent Document 8]
JP-B-63-28920 [Patent Document 9]
JP-A-4-229192 [Patent Document 10]
JP-A-63-35238 [Patent Document 11]
Japanese Patent Publication No. 11-502410 [Patent Document 12]
JP-A-8-248028 [Patent Document 13]
JP-A-10-179193 [Patent Document 14]
JP 2000-7696 A
[Problems to be solved by the invention]
The problem to be solved by the present invention is a method for stabilizing a nucleoside or a nucleotide or a derivative thereof in a solution having stability that can withstand a long-term stable liquid reagent and maintaining good reactivity with various samples and It is to provide the composition.
[0007]
[Means for Solving the Problems]
The present inventors have conducted various studies to achieve the above object, and as a result, by coexisting an antibiotic in a solution containing a nucleoside or nucleotide or a derivative thereof and a substance that degrades a nucleoside or nucleotide or a derivative thereof as a contaminant substance. The present inventors have found that a nucleoside or a nucleotide or a derivative thereof in a solution is kept stable in a liquid state for a long period of time, and that good reactivity with various samples is maintained. That is, the present invention has the following configuration.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
As one embodiment of the present invention, a neutral fat measurement reagent comprising a two-reagent system containing glycerol kinase, glycerophosphate oxidase, ATP, and the like as a first reagent and lipase, peroxidase, and the like as a second reagent, comprises: There is a method for stabilizing ATP, which comprises coexisting an antibiotic in a reagent or reaction solution containing the ATP.
[0009]
Further, as one embodiment of the present invention, an inorganic phosphorus measuring reagent comprising a two-reagent system containing xanthine oxidase, peroxidase, and the like as a first reagent and xanthine oxidase, purine nucleoside phosphorylase, peroxidase, inosine, and the like as a second reagent , There is also a method for stabilizing inosine, which comprises coexisting an antibiotic in a reagent or reaction solution containing inosine.
[0010]
In the present invention, "stable" means that the concentration of a nucleoside or a nucleotide or a derivative thereof is maintained at 2 to 35C, and specifically, 50 mM PIPES (pH 7.0) containing an antibiotic. When coexisting with 1 mM ATP in the buffer, the ATP residual concentration after storage at 35 ° C. for 2 weeks is 70% or more (preferably 80% or more, more preferably 90% or more) as compared with the concentration immediately after preparation. ) Means being maintained. When 12 mM inosine was co-present in a 20 mM MES (pH 6.8) buffer containing an antibiotic, the residual inosine concentration after storage at 35 ° C for 2 weeks was 70% lower than that immediately after preparation. % (Preferably 80% or more, more preferably 90% or more).
[0011]
In the present invention, “sufficient reactivity” refers to a neutral fat concentration in the range of 3000 mg / dL or less in the measurement of neutral fat, where the neutral fat concentration calculated using the measured absorbance of purified water and 200 mg / dL triolein solution as a control. It means recovering the theoretical value, specifically, the measured value does not fall below 95% of the theoretical value. In addition, in the measurement of inorganic phosphorus, it means that an inorganic phosphorus concentration calculated using the measured absorbance of purified water and a 5 mg / dL inorganic phosphorus solution as a control is a theoretical value within a range of 40 mg / dL or less. Means that the measured value does not fall below 95% of the theoretical value.
[0012]
The nucleoside or nucleotide or a derivative thereof of the present invention is not particularly limited. For example, the nucleoside or nucleotide as a coenzyme or a substrate in a measurement method using an enzyme of a biologically active substance in a biological component currently used for clinical diagnosis. Examples include reduced or oxidized NADH, reduced or oxidized NADPH, ATP, ADP, flavin adenine dinucleotide (FAD), inosine, xanthosine, pyridoxal pentaphosphate, and the like. Also included are polynucleotides used as probes or primers for gene amplification in the detection of nucleic acids in biological samples, as well as products of gene amplification reactions.
[0013]
The present invention also provides a nucleoside or a nucleotide or a derivative thereof in a solution containing a substance that degrades a nucleoside or a nucleotide or a derivative thereof, wherein an antibiotic is coexistent with a solution containing the nucleoside or the nucleotide or a derivative thereof. It is a method of stabilizing.
[0014]
The substances of the present invention that degrade nucleosides or nucleotides or derivatives thereof include (1) substances that coexist with nucleosides or nucleotides or derivatives thereof under storage conditions, and (2) substances that do not coexist with nucleosides or nucleotides or derivatives thereof under storage conditions. Substances that decompose nucleosides or nucleotides or derivatives thereof contained in substances coexisting during the reaction and (3) biological samples such as blood and urine to be measured are included.
[0015]
Specifically, for example, nucleotide pyrophosphatase, adenosine triphosphatase, ribonuclease, deoxyribonuclease, nucleosidase, nucleotidase and the like can be used to degrade nucleic acids by enzymatic action. Although it is considered that the optimum pH of the decomposition reaction and the conditions for activating the decomposition reaction are different depending on the origin, they can be appropriately optimized by changing the combination of the kind and the concentration of the antibiotic of the present invention.
[0016]
The substance that degrades the nucleoside or nucleotide or a derivative thereof of the present invention can be detected, for example, as follows.
(Detection method 1)
{Circle around (1)} Test solution acetate buffer (pH 6.0) 75 mM
p-Nitrophenyl phosphate (PNPP) 5 mM
(2) Measurement 1.5 mL of the test solution was incubated at 30 ° C, and 0.5 mL of the test sample was added and mixed. The mixture was reacted at 30 ° C for exactly 15 minutes, and 2.0 mL of 0.2 M sodium carbonate solution was added. And stop the reaction. Thereafter, absorbance at 400 nm is measured using purified water as a control. As a reagent blank, 2.0 mL of a 0.2 M sodium carbonate solution is added to 1.5 mL of the measurement test solution, reacted at 30 ° C. for 15 minutes, and 0.5 mL of a test sample is added and mixed, and the absorbance at 400 nm is measured in the same manner. The activity of decomposing 1 micromol of PNPP per minute was defined as 1 U, and the absorbance obtained was expressed by the formula: (absorbance of test sample−absorbance of reagent blank) × 4 / (18.1 × 15 × 0). .5).
[0017]
(Detection method 2)
(1) Tris-HCl buffer (pH 7.5) 100 mM
ATP 2.4 mM
Phosphoenolpyruvate 0.87 mM
KCl 10 mM
MgSO ₄ 2.5 mM
NADH 0.15 mM
Pyruvate kinase 10U / mL
Lactate dehydrogenase 10U / mL
(2) Measurement 2.6 mL of the test solution was incubated at 25 ° C, 0.2 mL of the test sample was added and mixed, and the change in absorbance at 340 nm at 25 ° C was monitored for 3 to 4 minutes using purified water as a control. From the time course, the rate of change in absorbance per minute of the initial linear portion is determined. A reagent blank is similarly performed using purified water instead of the test sample. The activity of oxidizing 1 micromol of NADH per minute was defined as 1 U, and the absorbance obtained was expressed by the formula: (change rate of absorbance of test sample−change rate of absorbance of reagent blank) × 2.8 / (6 .22 × 0.2).
[0018]
(Detection method 3)
(1) Measurement reagent solution Measurement reagent solution 1
Tris-HCl buffer (pH 7.5) 50 mM
Inosine 0.2 mM
Purine nucleoside phosphorylase 2U / mL
Xanthine oxidase 10U / mL
Measurement reagent 2
Tris-HCl buffer (pH 7.5) 50 mM
Inosine 20 mM
{Circle around (2)} Measurement solution 1 Add 2.5 mL of test sample to 2.5 mL, mix and incubate at 37 ° C for 5 minutes, add 0.3 mL of test solution 2 and mix, and measure the change in absorbance at 293 nm at 37 ° C. The purified water is monitored as a control for about 20 minutes, and the rate of change in absorbance per 1 to 20 minutes after addition of the test solution 2 is determined. A reagent blank is similarly performed using purified water instead of the test sample. The decomposing activity was defined as 1 U, the activity of producing 1 micromolar uric acid per minute, and the obtained absorbance was expressed by the formula: (test sample absorbance change rate-reagent blank absorbance change rate) x 3 / (12.5 × 0.2).
[0019]
(Detection method 4)
{Circle around (1)} Reaction reagent PIPES buffer (pH 7.0) 100 mM
ATP 3mM
KCl 20 mM
MgSO ₄ 6 mM
(2) Measurement test solution PIPES buffer (pH 7.0) 50 mM
Glucose 100 mM
MgSO ₄ 5 mM
NAD 0.2 mM
Glucose-6-phosphate dehydrogenase 2U / mL
Hexokinase 2U / mL
Polyoxyethylene octyl phenyl ether 0.1%
{Circle around (3)} 2 mL of the test solution is mixed with 2 mL of the test sample and incubated at 35 ° C. for exactly 48 hours, and the ATP concentration is measured as the measurement target. As a control, the test sample is replaced with purified water. In these ATP measurements, 0.2 mL of the test sample is added to 3 mL of the test solution, mixed, incubated at 37 ° C. for 10 minutes, and the absorbance at 340 nm is measured using purified water as a control. The ATP concentration is calculated by performing calibration on the basis of the absorbance measured in the same manner for purified water and a solution with a known concentration of ATP instead of the test sample. The decomposition activity is calculated as the amount of ATP decrease at 35 ° C. for 48 hours.
[0020]
(Detection method 5)
{Circle around (1)} Reaction reagent PIPES buffer (pH 7.0) 100 mM
NAD 3mM
KCl 20 mM
MgSO ₄ 6 mM
(2) Measurement test solution PIPES buffer (pH 7.0) 50 mM
Glucose-6-phosphate 4 mM
Glucose-6-phosphate dehydrogenase 3U / mL
Polyoxyethylene octyl phenyl ether 0.1%
{Circle around (3)} 2 mL of the test solution is mixed with 2 mL of the test sample and incubated at 35 ° C. for exactly 48 hours, and the NAD concentration is measured. As a control, the test sample is replaced with purified water. In these NAD measurements, 0.2 mL of the test sample is added to 3 mL of the test solution, mixed, incubated at 37 ° C. for 10 minutes, and the absorbance at 340 nm is measured using purified water as a control. The NAD concentration is calculated by performing calibration on the basis of the absorbance measured in the same manner for purified water and a solution with a known concentration of ATP instead of the test sample. Degradation activity is calculated as the amount of NAD reduction at 35 ° C. for 48 hours.
[0021]
(Detection method 6)
{Circle around (1)} Reaction reagent MES buffer (pH 6.8) 20 mM
Inosine 12 mM
(2) Measurement solution phosphate buffer (pH 6.8) 50 mM
Purine nucleoside phosphorylase 2U / mL
Xanthine oxidase 10U / mL
4-aminoantipyrine 0.05%
ADPS 0.1%
Polyoxyethylene octyl phenyl ether 0.1%
{Circle around (3)} 2 mL of the reaction sample solution is mixed with 2 mL of the test sample and incubated at 35 ° C. for exactly 48 hours, and the inosine concentration is measured as a measurement target. As a control, the test sample is replaced with purified water. For the measurement of inosine, 0.1 mL of a test sample is added to 3 mL of the test solution, mixed, incubated at 37 ° C. for 10 minutes, and the absorbance at 340 nm is measured using purified water as a control. The inosine concentration is calculated by performing calibration on the basis of the absorbance measured in the same manner for purified water and a solution of known inosine concentration instead of the test sample. Degradation activity is calculated as the amount of inosine decrease at 35 ° C. for 48 hours.
[0022]
The concentration (activity) of the substance that degrades a nucleoside or nucleotide or a derivative thereof in the solution of the present invention is not particularly limited, as long as it is at least one that is significantly detected by the above-described detection methods 1 to 6. The effect has an effect on substances that degrade these nucleosides or nucleotides or derivatives thereof.
[0023]
The antibiotic of the present invention is not particularly limited as long as it has an effect of stabilizing a nucleoside or a nucleotide or a derivative thereof, but is preferably an aminoglycoside antibiotic or a chloramphenicol antibiotic or a macrolide antibiotic or these. Are used. Examples of aminoglycoside antibiotics include streptomycin, kanamycin, gentamicin, amikacin, tobramycin, fradiomycin, bekanamycin, dibekacin, sisomycin, micronomycin, netilmycin astromycin, isepamicin, arbekacin and the like. Chloramphenicol antibiotics include chloramphenicol. Macrolide antibiotics include erythromycin, clarithromycin, roxithromycin and the like.
[0024]
These concentrations need to be those necessary for stabilizing the nucleoside or nucleotide or its derivative, but in consideration of their solubility, 0.005 to 0.2%, preferably 0.005 to 0.1%. Used in the range.
[0025]
The buffer used in the present invention includes Tris buffer, phosphate buffer, GOOD buffer and the like. Examples of the GOOD buffer include MES, Bis-Tris, ACES, BES, MOPS, PIPES, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS. The concentration of the buffer is not particularly limited, and is used at 10 to 300 mM, preferably 50 to 200 mM. The pH of the buffer varies depending on the reaction conditions and the like to which the present invention is applied, but is adjusted in the range of 5 to 9 in consideration of the pH optimum for nucleosides or nucleotides or derivatives thereof. Further, 6 to 8.5 is preferable. Depending on the type of buffer or its pH, a substance that degrades nucleosides or nucleotides or derivatives thereof may be activated or inhibited.However, by appropriately adjusting the concentration of the antibiotic of the present invention, various buffers can be used. The type, concentration, and pH conditions can be selected.
[0026]
Preservatives other than antibiotics, surfactants, enzymes, substrates and the like may be further added to the solution of the present invention.
Preservatives include azide, 5-chloro-2-methyl-4-isothiazolin-3-one, imidazolidinyl urea, and the like. Examples of the surfactant include a nonionic surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant. These surfactants can be used alone or in combination of two or more kinds or other surfactants. In addition, depending on the type of surfactant, a substance that degrades nucleosides or nucleotides or derivatives thereof may be activated or inhibited, but various surfactants may be obtained by appropriately adjusting the concentration of the antibiotic of the present invention. It is possible to apply The concentration in the solution is not particularly limited, but is preferably in the range of 0.01% to 0.5%, and more preferably 0.02% to 0.2% has an influence on the enzyme reaction. It is preferable because it has few points. Among them, 0.05% to 0.15% is preferable. Enzymes include those obtained from mammals, plants, microorganisms, and the like, and those produced from genetically modified microorganisms in which these genes have been incorporated into other microorganisms. Contains In addition, for the purpose of improving the specificity and stability of these enzymes or modifying the property of adsorbing to the reaction cell, a group containing polyethylene glycol as a main component, a monosaccharide, a water-soluble oligosaccharide residue, a sulfopropyl group, etc. The above-mentioned enzyme may be chemically modified.
[0027]
The concentration of the nucleoside or nucleotide or its derivative in the solution of the present invention is not particularly limited.
[0028]
【Example】
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 following neutral fat measuring reagent containing 1 mM adenosine triphosphate (manufactured by Roche) and 0.05% of various antibiotics was added and dissolved in the first reagent solution. The solution was stored at 35 ° C. for 14 days. (The ratio of the concentration after storage to the concentration immediately after). In addition, a sample having a neutral fat concentration of 2000 mg / dL was measured using a reagent stored at 35 ° C. for 14 days, and the recovery of each measured value with respect to the theoretical value was calculated.
[0029]
(Preparation of reagent)
A neutral fat measuring reagent having the following composition was prepared.
First reagent PIPES-NaOH 50 mM pH 7.0
MgCl2 0.2g / L
EDTA · 2Na 0.3 mM
Adenosine triphosphate 1 mM
Emulgen A60 4g / L
Triton X-100 2g / L
4-aminoantipyrine 0.1 g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Glycerol kinase (GYK-311 manufactured by Toyobo) 3 U / 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
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
Triton X-100 1g / L
[0030]
(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.
[0031]
[Table 1]

[0032]
Results are shown in Table 1. In all of the examples, the ATP residual ratio was higher than that of the comparative example, indicating good stability, and the measured value recovery rate of neutral fat after storage was also good, and the reactivity was maintained.
[0033]
(Example 2)
Neutral fat measurement reagent described in Example 1 containing 1 mM of ATP · 2Na (manufactured by Roche). Kanamycin 0.005% to 0.2% was added to the first test solution, dissolved and stored at 35 ° C. for 14 days. The residual amount (the ratio of the concentration after storage to the concentration immediately after dissolution) was examined.
[0034]
[Table 2]

[0035]
Results are shown in Table 2. In all of the examples, the ATP residual ratio was higher than that of the comparative example, indicating good stability, and the measured value recovery rate of neutral fat after storage was also good, and the reactivity was maintained. The effect was confirmed even with a low concentration of kanamycin.
[0036]
(Example 3)
The following inorganic phosphorus measurement reagent containing 12 mM of inosine (Kojin Co., Ltd.) was added to each of the following two test solutions, and 0.05% of each antibiotic was dissolved. The solution was stored at 35 ° C. for 14 days. Was determined. In addition, a sample having an inorganic phosphorus concentration of 40 mg / dL was measured using a reagent stored at 35 ° C. for 14 days, and the recovery of each measured value with respect to the theoretical value was calculated.
[0037]
(Preparation of reagent)
An inorganic phosphorus measuring reagent having the following composition was prepared.
First reagent PIPES-NaOH 50 mM pH 7.0
EDTA · 2Na 0.3 mM
Triton X-100 2g / L
ADPS 0.1g / L
Xanthine oxidase (Toyobo XTO-211) 5U / mL
Peroxidase (PEO-301 manufactured by Toyobo) 1 U / mL
Ascorbate oxidase (ASO-311 manufactured by Toyobo) 3 U / mL
Second reagent PIPES-NaOH 50 mM pH 7.0
EDTA · 2Na 0.3 mM
Triton X-100 2g / L
4-aminoantipyrine 0.3 g / L
Inosine 12 mM
Xanthine oxidase (XTO-211 manufactured by Toyobo) 1 U / mL
Peroxidase (PEO-301 manufactured by Toyobo) 1 U / mL
Purine nucleotide phosphorylase (PNP-301 manufactured by Toyobo) 1 U / mL
[0038]
(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 5 mg / dL aqueous solution of inorganic phosphorus, and determined as the inorganic phosphorus concentration.
[0039]
[Table 3]

[0040]
Results are shown in Table 3. In each of the examples, the inosine residual ratio was higher than that of the comparative example, indicating good stability, and the measured value recovery rate of the inorganic phosphorus after storage was also good, and the reactivity was maintained.
[0041]
(Example 4)
The following neutral fat measurement reagent containing 1 mM adenosine triphosphate (manufactured by Roche) and 0.03 and 0.06% of kanamycin was added and dissolved in the first reagent solution, stored at 10 ° C. for 12 months, and the amount of ATP remaining (Ratio of the concentration after storage to the concentration immediately after dissolution) was examined. In addition, a sample having a neutral fat concentration of 2000 mg / dL was measured using a reagent stored at 10 ° C. for 12 months, and the recovery of each measured value with respect to the theoretical value was calculated.
[0042]
[Table 4]

[0043]
Results are shown in Table 4. In each of the examples, the ATP residual rate was higher than that of the comparative example, indicating good stability, and the measured value recovery rate of neutral fat after storage was also good, and the reactivity was maintained. The long-term storage stability was also good.
[0044]
【The invention's effect】
In the present invention, by coexisting an antibiotic in a solution containing a nucleoside or a nucleotide or a derivative thereof, the nucleoside or the nucleotide or the derivative thereof in the solution is kept stable in a liquid state for a long time, and is excellent for various samples. And a method for maintaining high reactivity.

Claims (4)

A method for stabilizing a nucleoside, a nucleotide, or a derivative thereof in a solution, comprising causing an antibiotic to coexist in a solution containing the nucleoside, a nucleotide, or a derivative thereof. The stabilizing method according to claim 1, wherein the antibiotic is an aminoglycoside antibiotic and / or a chloramphenicol antibiotic and / or a macrolide antibiotic. A stabilized composition of a nucleoside or a nucleotide or a derivative thereof in a solution, characterized in that a nucleoside or a nucleotide or a derivative thereof and an antibiotic are allowed to coexist. The stabilized composition according to claim 3, wherein the antibiotic is an aminoglycoside antibiotic and / or a chloramphenicol antibiotic and / or a macrolide antibiotic.