JP6487711B2 - Novel measurement method and composition for orthophosphate, alkaline phosphatase, pyrophosphate and the like using purine nucleoside phosphorylase - Google Patents

Description

  The present invention relates to a novel method for measuring orthophosphate, alkaline phosphatase, pyrophosphate, and the like by an enzyme cycling method using purine nucleoside phosphorylase, and a composition thereof.

  Alkaline phosphatase (ALP) is an enzyme that hydrolyzes a phosphate ester compound under alkaline conditions. ALP is one of the most important indicators of liver function in clinical tests. In addition, since ALP has a high specific activity, bovine ALP, for example, is used as a labeling enzyme for enzyme immunoassay. In a conventional method for measuring alkaline phosphatase, for example, p-nitrophenyl phosphate is used as a substrate, and p-nitrophenol hydrolyzed and released has an absorption peak in the vicinity of 405 nm under alkalinity. A method of detecting ALP as a labeling enzyme with high sensitivity by NAAD cycling using NADP as a substrate has also been reported (Non-patent Document 1: Masakazu Koda et al., Instruments and Reagents, 21, 633 (1998)). As a more sensitive detection method, a chemiluminescent substrate, for example, 3- (2′-spiroadamantane) -4-methoxy-4- (3 “-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) is used. A method is known (Non-patent Document 2: Clin Chem 35: 1441-1446 (1989)).

  Pyrophosphate (PPi) is a diphosphate molecule and is used as a substrate or product in a wide variety of enzymatic reactions. Since pyrophosphate is a by-product in PCR, which is a nucleic acid amplification reaction, the measurement is useful for detecting and quantifying nucleic acids. Conventionally, as a method for measuring pyrophosphate, a method using pyruvate phosphate dikinase and luciferase (Patent Document 1: Japanese Patent Application Laid-Open No. 2007-097471), a method using hypoxanthine phosphoribosyltransferase, xanthine dehydrogenase / oxidase ( Patent Document 2: JP 2003-174900 A), a method using a kinase, an enzyme that generates ATP from pyrophosphate, and a dehydrogenase that uses NAD (P) as a coenzyme (Patent Document 3: JP 2006-187251 A) Alternatively, a method using pyruvate phosphate dikinase, nicotinamide nucleotide adenyl transferase, and dehydrogenase (Patent Document 4: JP 2009-225784 A) is known.

  Orthophosphoric acid (Pi) is a monophosphate molecule, and is important as an indicator of hyperphosphatemia and the like as inorganic phosphorus in clinical examination items. As a conventional method for measuring orthophosphoric acid, a phosphomolybdic acid reduction method is known as a chemical method, and as a method using an enzyme, for example, a method using sucrose phosphorylase (Patent Document 5: JP-A 63-63). No. 49100), a method using maltose phosphorylase, and the like are known. Furthermore, purine nucleoside phosphorylase (PNPL) is combined with xanthine oxidase to reduce the amount of hydrogen peroxide produced (Non-patent Document 3: Anal Biochem 222: 168-175, 1994), and also reduced using xanthine dehydrogenase. A method for quantifying type NAD has been reported (Patent Document 6: Japanese Patent Laid-Open No. 6-13837). In addition, as a highly sensitive measurement method, the following method using an enzyme cycling reaction has been reported. That is, a method using an immobilized enzyme reactor based on an enzyme cycling reaction combining PNPL and alkaline phosphatase (Non-patent Document 4: Analytical Chimica Acta 238: 339-342, 1990), glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) (EC 1.2.1.12) has been reported (Patent Document 7: JP-A-4-349898).

JP 2007-097471 A JP 2003-174900 A JP 2006-187251 A JP 2009-225784 A JP 63-49100 A JP-A-6-1113837 JP-A-4-349898

Masakazu Koda et al., Equipment and Reagents, 21, 633 (1998) Clin Chem 35: 1441-1446 (1989) Anal Biochem 222: 168-175, 1994 Analytical Chimica Acta 238: 339-342, 1990

  Conventional measuring methods for orthophosphoric acid, alkaline phosphatase, pyrophosphoric acid and the like have a drawback that a highly sensitive method has a complicated operation, and a method that has a simple operation has a low sensitivity. Therefore, an object of the present invention is to provide a measuring method for orthophosphoric acid, alkaline phosphatase, pyrophosphoric acid and the like, and a composition for the measurement, which has higher sensitivity than conventional methods and is easy to operate. .

  As described above, various measuring methods for orthophosphoric acid, alkaline phosphatase, pyrophosphoric acid and the like have been conventionally known. Among these, examples of methods that can be measured with high sensitivity include the methods disclosed in Non-Patent Document 1, Patent Document 1, and Non-Patent Document 4. However, these methods are complicated in operation, and a dedicated device such as a fluorometer may be required. On the other hand, examples of a simple measurement method include methods disclosed in Non-Patent Document 3 and Patent Document 6. However, none of these methods can measure with high sensitivity.

  In these circumstances, the present inventors tried to measure by enzyme cycling method using the enzymatic properties of purine nucleoside phosphorylase, and surprisingly, pyrophosphate, alkaline phosphatase, and orthophosphoric acid were used. The present invention has been completed by discovering that it is possible to easily measure the above and the like with high sensitivity.

The present invention has the following configuration.
[1]
At least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate,
(1) Purine nucleoside phosphorylase, which catalyzes a forward reaction for producing ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphoric acid in the presence of purine nucleoside, and a reverse reaction thereof. A purine nucleoside phosphorylase that can utilize at least different purines in the forward reaction and the reverse reaction, a purine nucleoside having a first purine, and a second purine different from the first purine are brought into contact with the sample. A step of causing a cycling reaction of the following formula (1):
(2) detecting a change amount of a signal corresponding to a change amount of at least one of a purine nucleoside having a first purine, a first purine, a second purine, and a purine nucleoside having a second purine; ,
(3) Based on the detected amount of change in signal, calculate the amount of at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate And a process of
Including
When the measurement object is alkaline phosphatase, the phosphate compound can be hydrolyzed with alkaline phosphatase in the presence of a phosphate ester compound that can produce orthophosphoric acid. The sample has been processed,
When the measurement target is pyrophosphoric acid, the sample is treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid.
Measuring method.

[1-1]
The method according to [1], wherein the measurement target is at least one of orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate.

[1-2]
The measurement object is alkaline phosphatase,
The sample is treated in the presence of a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphate, under conditions that allow the phosphate ester compound to be hydrolyzed to produce phosphate. The amount of alkaline phosphatase is calculated based on the amount of signal change
The method according to [1].

[1-3]
The measurement object is pyrophosphoric acid, and the sample is treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid,
Based on the amount of change in the detected signal, the amount of pyrophosphate is calculated.
The method according to [1].

[2]
Purine nucleoside phosphorylase is EC 2.4.2.1, which produces ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphate in the presence of a purine nucleoside having a first purine The method according to any one of [1] to [1-3], which is an enzyme that catalyzes a normal reaction and its reverse reaction and can use at least different purines in the normal reaction and the reverse reaction.

[3]
The measurement method of [1] or [2], wherein the step (2) is a step of detecting a change amount of a signal corresponding to an increase amount of a purine nucleoside having a first purine and / or a second purine .

[3-1]
In the above step (2), the first purine has substrate specificity, but the second purine has no substrate specificity, or the purine nucleoside having the first purine has substrate specificity. The detection enzyme having substrate specificity is used for the purine nucleoside having the second purine, but the amount of the purine nucleoside having the first purine and / or the second purine is increased. The method according to any one of [1] to [3], wherein a change amount of a signal to be detected is detected.

[3-2]
The above step (2) corresponds to the increased amount of the first purine using a detection enzyme having the substrate specificity for the first purine but not having the substrate specificity for the second purine. The method according to any one of [1] to [3-1], wherein the amount of change in signal is detected.

[4]
The combination of the first purine and the second purine different from the first purine is any two of adenine, guanine, hypoxanthine and xanthine, any one of [1] to [3-2] The method described in 1.

[4-1]
The combination of the first purine and the second purine different from the first purine is a combination of hypoxanthine and guanine, a combination of hypoxanthine and adenine, a combination of xanthine and guanine, or a combination of xanthine and adenine. The method according to any one of [1] to [4], which is a combination.

[4-2]
The method according to any one of [1] to [4-1], wherein the first purine is hypoxanthine or xanthine.

[4-3]
The method according to any one of [1] to [4-1], wherein the first purine is guanine.

[4-4]
The method according to any one of [1] to [4-1], wherein the first purine is adenine.

[5]
In the above step (2), using xanthine dehydrogenase having substrate specificity for hypoxanthine and / or xanthine, in the presence of thio-NAD or NAD, the amount of change in signal corresponding to the increased amount of hypoxanthine and / or xanthine is determined. The method according to [4-2], which is detected.

[5-1]
In the above step (2), using xanthine oxidase having substrate specificity for hypoxanthine and / or xanthine, the amount of change in signal corresponding to the increased amount of hypoxanthine and / or xanthine is detected [4-2] The method described in 1.

[5-2]
The method according to [4-3], wherein in the step (2), the amount of change in the signal corresponding to the increased amount of guanine is detected using guanine deaminase having substrate specificity for guanine.

[5-3]
The method according to [4-4], wherein in the step (2), an adenine deaminase having substrate specificity for adenine is used to detect a change in signal corresponding to the increased amount of adenine.

[6]
A method for measuring alkaline phosphatase, comprising:
(1) A purine nucleoside phosphorylase (EC 2.4.2.1), which generates ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphoric acid in the presence of inosine, And purine nucleoside phosphorylase that catalyzes the reverse reaction and can use guanine in the reverse reaction, inosine, and guanine are brought into contact with the sample, and the cycling reaction of the following formula (2) is reacted:
(2) using xanthine dehydronase, detecting a change amount of a signal corresponding to an increased amount of hypoxanthine;
(3) calculating the amount of alkaline phosphatase based on the amount of change in the detected signal;
Including
In the presence of a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphate, the sample is treated under conditions that allow the phosphate ester compound to be hydrolyzed to produce phosphate.
The method according to any one of [1], [1-2], [2] to [4-2], and [5].

[7]
A measurement composition for measuring at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate,
(A) a purine nucleoside phosphorylase, a positive reaction for producing ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphate in the presence of a purine nucleoside having a first purine, and A purine nucleoside phosphorylase that catalyzes a reverse reaction and can use at least different purines in the forward and reverse reactions,
(B) a purine nucleoside having a first purine;
(C) a second pudding different from the first pudding;
A composition comprising:
A composition in contact with the sample,
When the measurement object is alkaline phosphatase, the phosphate compound can be hydrolyzed with alkaline phosphatase in the presence of a phosphate ester compound that can produce orthophosphoric acid. The sample has been processed,
When the measurement target is pyrophosphoric acid, the sample is processed under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid.
Composition.

[7-1]
The composition according to [7], wherein the measurement target is orthophosphoric acid, ribose-1-phosphate, and / or deoxyribose-1-phosphate.

[7-2]
The measurement object is alkaline phosphatase,
In the presence of a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphate, it is contacted with a sample that has been treated under conditions that allow the phosphate ester compound to be hydrolyzed to produce phosphate. [7] The composition according to [7].

[7-3]
The composition according to [7], wherein the measurement target is pyrophosphoric acid, and the sample is contacted with a sample that has been treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid.

[8]
Purine nucleoside phosphorylase is EC 2.4.2.1, which produces ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphate in the presence of a purine nucleoside having a first purine The composition according to any one of [7] to [7-3], which catalyzes a normal reaction and a reverse reaction thereof and can use at least different purines in the normal reaction and the reverse reaction.

[8-1]
The first purine has substrate specificity, but the second purine has no substrate specificity, or the purine nucleoside having the first purine has no substrate specificity. The composition according to any one of [7] to [8], wherein the purine nucleoside having the second purine further comprises a detection enzyme having substrate specificity.

[8-2]
The composition according to any one of [7] to [8-1], wherein the first purine contains a detection enzyme that has substrate specificity but the second purine does not have substrate specificity.

[9]
The combination of the first purine and the second purine different from the first purine is any two of adenine, guanine, hypoxanthine, and xanthine, [7] to [8-2] The composition in any one of.

[9-1]
The combination of the first purine and the second purine different from the first purine is a combination of hypoxanthine and guanine, a combination of hypoxanthine and adenine, a combination of xanthine and guanine, or a combination of xanthine and adenine. The composition according to any one of [7] to [9], which is a combination.

[9-2]
The composition according to any one of [7] to [9-1], wherein the first purine is hypoxanthine or xanthine.

[9-3]
The composition according to any one of [7] to [9-1], wherein the first purine is guanine.

[9-4]
The composition according to any one of [7] to [9-1], wherein the first purine is adenine.

[10]
Xanthine dehydrogenase having substrate specificity for hypoxanthine and / or xanthine;
NAD or thio-NAD,
The composition according to [9-2], comprising:

[10-1]
The composition according to [9-2], comprising hypoxanthine and / or xanthine oxidase having substrate specificity for xanthine.

[10-2]
The composition according to [9-3], comprising guanine deaminase having substrate specificity for guanine.

[10-3]
The composition according to [9-4], comprising adenine deaminase having substrate specificity for adenine.

[11]
A measurement composition for measuring alkaline phosphatase,
(A) a purine nucleoside phosphorylase, which catalyzes a forward reaction for producing ribose-1-phosphate from orthophosphoric acid in the presence of inosine, and its reverse reaction, and can use guanine in the reverse reaction;
(B) inosine;
(C) guanine,
(D) xanthine dehydrogenase;
(E) NAD or thio DNA;
A composition comprising:
Used for samples treated with phosphate ester compounds that can be hydrolyzed with alkaline phosphatase to produce orthophosphoric acid,
[7] The composition according to any one of [7-2], [8] to [9-2], and [10].

[12]
[7] A kit comprising the composition according to any one of [11].

  INDUSTRIAL APPLICABILITY According to the present invention, a measurement method and a composition for measurement capable of easily and easily measuring pyrophosphate, alkaline phosphatase, orthophosphate, and the like by an enzyme cycling method using purine nucleoside phosphorylase. As well as kits for measurement.

2 is a graph showing the concentration of dipotassium hydrogen phosphate in the sample and the amount of change in absorbance in Example 1. FIG. 2 is a graph showing the concentration of dipotassium hydrogen phosphate in the sample and the amount of change in absorbance in Example 2. 6 is a graph showing the concentration of dipotassium hydrogen phosphate in the sample and the amount of change in absorbance in Example 4. 6 is a graph showing the concentration of dipotassium hydrogen phosphate in the sample and the amount of change in absorbance in Example 5. 6 is a graph showing ALP activity and absorbance change amount in a sample in Example 6. 6 is a graph showing the sodium pyrophosphate concentration in the sample and the amount of change in absorbance in Example 7. 6 is a graph showing the concentration of dipotassium hydrogen phosphate in the sample and the amount of change in absorbance in Example 8.

  Hereinafter, an embodiment of the present invention (hereinafter also referred to as “the present embodiment”) will be specifically described. However, it should not be understood that the following embodiments limit the present invention. From this disclosure, various alternative embodiments, examples, and operation techniques should be apparent to those skilled in the art. It should be understood that the present invention includes various embodiments and the like not described herein. The EC number (Enzyme Commission numbers) in this specification is a number confirmed as of February 18, 2014.

The method according to the present embodiment is a method for measuring at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate,
(1) A purine nucleoside phosphorylase, a positive reaction for producing ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphoric acid in the presence of a purine nucleoside having a first purine, and A purine nucleoside phosphorylase that catalyzes a reverse reaction and can use at least different purines in the forward reaction and the reverse reaction, a purine nucleoside having a first purine, and a second purine different from the first purine. , In contact with a sample and reacting a cycling reaction of the following formula (3):
(2) detecting a change amount of a signal corresponding to a change amount of at least one of a purine nucleoside having a first purine, a first purine, a second purine, and a purine nucleoside having a second purine; ,
(3) Based on the amount of change in the detected signal, calculate the amount of at least one of orthophosphate, ribose-1-phosphate, deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate. Process,
Including
When the measurement object is alkaline phosphatase, the condition in which the phosphate ester compound can be hydrolyzed to produce phosphate in the presence of the phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphate The sample has been processed at
When the measurement target is pyrophosphoric acid, the sample is treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid.
This is a measurement method.

  The measurement object in the method according to the present embodiment is at least one of orthophosphate, ribose-1-phosphate and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate. Preferable measurement objects include alkaline phosphatase or pyrophosphate, and more preferably alkaline phosphatase. Orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate can be measured separately or simultaneously. When orthophosphoric acid, ribose-1-phosphate, and / or deoxyribose-1-phosphate are mixed in a sample, when it is desired to quantify only one or two kinds, it is known prior to the enzyme cycling reaction. Using this method, the non-measurement target may be converted into another substance.

  The purine nucleoside phosphorylase in the method according to the present embodiment is a positive reaction that generates ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphate in the presence of a purine nucleoside having a first purine. And an enzyme that catalyzes the reverse reaction and can use at least different purines in the forward reaction and the reverse reaction, respectively. Confirm whether it catalyzes the forward reaction and the reverse reaction of producing ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphoric acid in the presence of a purine nucleoside having a first purine As a method, for example, several methods for detecting known purine nucleosides and purine bases by high performance liquid chromatography (HPLC) have been reported, and it is possible to confirm the reaction product using these methods. is there. Further, orthophosphoric acid as a product of the reverse reaction can be confirmed using an enzyme other than purine nucleoside phosphorylase. For example, orthophosphate produced by the reverse reaction of purine nucleoside phosphorylase in the presence of hypoxanthine and ribose-1-phosphate is converted to β-glucose-1-phosphate by maltose phosphorylase in the presence of maltose. Furthermore, it can be easily quantified by combining β-phosphoglucomutase and glucose-6-phosphate dehydrogenase, but is not limited thereto. In addition, examples of the method for confirming whether different purines can be used in the normal reaction and the reverse reaction include, but are not limited to, a method such as the detection method by the HPLC method described above.

  The purine nucleoside phosphorylase in the method according to the present embodiment is not particularly limited, and examples thereof include a purine nucleoside phosphorylase whose EC number (Enzyme Commission number) is specified by EC 2.4.2.1. Purine nucleoside phosphorylase in the method according to the present embodiment widely exists from higher animals to microorganisms, and its origin is not particularly limited. For example, it can be obtained from Bacillus stearothermophilus as a microorganism-derived enzyme. In addition, microorganism-derived enzymes are commercially available from Toyobo Co., Ltd., and Bacillus-derived enzymes are commercially available from Asahi Kasei Pharma Corporation. Conventional methods may be used for culturing these derived cells and tissues and isolating and purifying the enzyme. For example, after culturing a microorganism, a product having a purity increased to a practical level can be obtained by combining known purification methods such as column chromatography. It can also be obtained by culturing and using transformed cells such as E. coli into which the gene has been incorporated. Furthermore, the purine nucleoside phosphorylase according to the present embodiment may be genetically modified for the purpose of improving enzyme properties. For example, a gene encoding the enzyme protein can be isolated by a conventional method and obtained by a method of expressing in a suitable host using a gene recombination technique.

  “Purine” in the method according to the present embodiment means not only a naturally occurring purine but also a derivative or a similar substance synthesized chemically or enzymatically.

  As the first purine in the method according to the present embodiment, for example, adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine, 7-methylguanine, 7-methyladenine, 7-methylhypoxanthine, Examples include 2-amino-6-mercapto-7-methylpurine, 8-azahypoxanthine, 8-guanine, and derivatives thereof, but are not limited thereto. Preferably, adenine, guanine, hypoxanthine, and xanthine are mentioned. More preferably, hypoxanthine and xanthine are mentioned.

  The “purine nucleoside” in the method according to the present embodiment is a binding substance between the above purine and ribose, or a binding substance between the above purine and deoxyribose, and is not limited to a naturally occurring purine nucleoside. It is meant to include its derivatives and similar substances synthesized chemically or enzymatically. Examples of the purine nucleoside having the first purine include, but are not limited to, adenosine, guanosine, inosine, xanthosine, deoxyadenine, deoxyguanine, deoxyinosine, and deoxyxanthosine. Inosine and xanthosine are preferable.

  Examples of the second purine different from the first purine in the method according to the present embodiment include adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine, 7-methylguanine, 7- Examples include methyladenine, 7-methylhypoxanthine, 2-amino-6-mercapto-7-methylpurine, 8-azahypoxanthine, and 8-guanine, which are not selected as the first purine. However, it is not limited to these. Preferably, adenine, guanine, hypoxanthine, xanthine, etc. are mentioned, More preferably, adenine, guanine, etc. are mentioned.

  Examples of the purine nucleoside having the second purine include, but are not limited to, adenosine, guanosine, inosine, xanthosine, deoxyadenine, deoxyguanine, deoxyinosine, and deoxyxanthosine. Adenosine and guanosine are preferable.

In the method according to the present embodiment, the combination of the first purine and the second purine is not particularly limited, but may be a combination of any two of adenine, guanine, hypoxanthine and xanthine, for example. Preferably, a combination of hypoxanthine and guanine, a combination of hypoxanthine and adenine, a combination of xanthine and guanine, and a combination of xanthine and adenine. An example of the cycling reaction in the combination of hypoxanthine and guanine is shown by the following formula (4).

  The sample in the method according to the present embodiment is not particularly limited, but is, for example, a human or animal biological sample, preferably human blood, more preferably human serum. Whether or not the sample contains at least one of orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; It may be unknown before being performed, and the method according to the present embodiment is performed so that the sample is at least one of orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate; It may be apparent that it does not contain phosphatase; or pyrophosphate.

  When the measurement target is alkaline phosphatase, a sample after adding a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to the sample and hydrolyzing the phosphate ester compound to obtain orthophosphoric acid is used. The phosphate compound is not particularly limited as long as it can hydrolyze alkaline phosphatase. For example, β-glycerophosphate, α-glycerophosphate, phenylphosphate, p-nitrophenylphosphate, phosphorylcholine, and nicotine Amidoadenine dinucleotide phosphate (NADP) and the like can be mentioned. P-nitrophenyl phosphate is preferred. As a method for hydrolyzing the phosphate ester compound, a known method may be used. For example, a phosphoric acid ester compound as a substrate may be treated with alkaline phosphatase that can be contained in a sample at a certain temperature, for example, 37 ° C. for a certain time. Specifically, the enzyme treatment is performed in the presence of the phosphate ester compound using a buffer solution having a buffer region on the alkali side. When alkaline phosphatase is not included in the sample, orthophosphoric acid is not produced in the sample even if a phosphate ester compound that can be hydrolyzed with alkaline phosphatase is added to the sample.

  When the measurement target is pyrophosphoric acid, a sample after treatment for hydrolyzing pyrophosphoric acid to obtain orthophosphoric acid is used. As the treatment for hydrolyzing pyrophosphoric acid to orthophosphoric acid, for example, a known chemical treatment using acid or alkali can be used, or enzymatic hydrolysis can be used. In the case of enzymatic treatment, for example, an enzyme reaction capable of converting known pyrophosphate into orthophosphate may be used. Specifically, pyrolysis of pyrophosphate by pyrophosphatase (EC 3.6.1.1), reverse reaction of pyruvate phosphate dikinase (EC 2.7.9.1), and the like can be used. Yeast-derived pyrophosphatase is commercially available. When pyrophosphate is not contained in the sample, orthophosphoric acid is not generated in the sample even if the sample is treated as described above.

  In the method according to the present embodiment, the cycling reaction is not limited to this. For example, a known method may be referred to. Specifically, an excess amount, for example, 0.1 to 12 mmol / L, preferably 0.2 to 6 mmol / L of the first purine nucleoside and the second purine are added to the reaction solution, and a pH buffer is added thereto. 20-200 mmol / L. The pH of the reaction solution may be appropriately selected as long as the reaction proceeds efficiently. Usually, the pH may be adjusted to 5.5 to 8.5. The cycling reaction may be started by adding the sample and purine nucleoside phosphorylase to the reaction solution thus adjusted. Although reaction temperature is not specifically limited, For example, it is 25-42 degreeC, Preferably it is 30-37 degreeC.

  In the method according to the present embodiment, in order to cause a cycling reaction, the amount of purine nucleoside phosphorylase to be added to the reaction solution is necessary, for example, by calculating the cycling rate with reference to a known method and considering the reaction time. The amount of enzyme can be determined. The amount of purine nucleoside phosphorylase is, for example, 0.5 to 1000 u / mL, preferably 2 to 250 u / mL, but is not limited thereto. When the amount of the purine nucleoside phosphorylase exceeds 1000 u / mL, the binding between the purine nucleoside phosphorylase and the substrate increases, and amplification may reach its peak.

  In the method according to the present embodiment, a signal corresponding to a change amount of at least one of a purine nucleoside having a first purine, a first purine, a second purine, and a purine nucleoside having a second purine is detected. In this step, the amount of change in the signal corresponding to the increased amount of the purine nucleoside having the first purine and / or the second purine is preferably detected, but is not limited thereto.

  As a step of detecting the amount of change in signal, known HPLC methods, enzyme methods, and the like can be used, but are not limited thereto. When using the HPLC method, a chelating agent or the like is added to the reaction solution to terminate the enzyme reaction, and then a reverse-phase column or the like is appropriately selected to prepare a purine having the first purine and / or the second purine. What is necessary is just to quantify a nucleoside. Information on these HPLC analysis methods is readily available from resin manufacturers. Further, for example, the amount of change in signal can be detected by an enzymatic method. Specifically, the first purine has substrate specificity, but the second purine has no substrate specificity, or the purine nucleoside having the first purine has substrate specificity. However, a detection enzyme having substrate specificity is used for the purine nucleoside having the second purine, and the signal corresponding to the increased amount of the purine nucleoside having the first purine and / or the second purine is used. A method for detecting the amount of change is an enzymatic method. Preferably, the first purine has substrate specificity, but the second purine has no substrate specificity, and the amount of change in signal corresponding to the increased amount of the first purine is detected using a detection enzyme. A method for detecting the above is mentioned as an enzymatic method.

  More preferably, xanthine dehydrogenase (EC 1.2.1.37) is used to increase hypoxanthine and / or xanthine in the presence of thionicotinamide adenine dinucleotide (thioNAD) or nicotinamide adenine dinucleotide (NAD). A method for detecting a change in signal corresponding to the amount, a method for detecting a change in signal corresponding to an increase in hypoxanthine and / or xanthine using xanthine oxidase (EC1.17.3. 2), adenine Using deaminase (EC 3.5.4.2), a method for detecting a change in signal corresponding to the increased amount of adenine, and using guanine deaminase (EC 3.5.4.3), The method of detecting the amount of change in the signal corresponding to the increase amount, etc. It is not limited. Among these detection enzymes, xanthine dehydrogenase and xanthine oxidase are commercially available and easily available. In addition, since adenine deaminase and guanine deaminase are known to exist in bacteria and the like, they can be easily obtained by culturing and purifying according to conventional methods with reference to the literature on these. Furthermore, when the gene sequences of the enzymes that produce these are known, the genes can be obtained from cDNA or synthesis and produced as recombinants. For example, a paper on the expression of guanine deaminase derived from E. coli (J. Bacteriol 182, 4658-4660, 2000) has been reported.

  In order to specifically implement the enzymatic method for detecting the amount of change in signal, a known method may be used. As an example, when inosine or xanthosine is selected as the purine nucleoside with the first purine and adenine or guanine is selected as the second purine, the amount of change in signal corresponding to the increase in hypoxanthine and / or xanthine is detected. For this purpose, if xanthine dehydrogenase (Asahi Kasei Pharma Co., Ltd., T134) is used, adenine or guanine is not detected, but only hypoxanthine or xanthine, which is a positive reaction change product of inosine or xanthosine, can be detected. Similarly, since xanthine oxidase commercially available from Toyobo Co., Ltd. does not act on adenine, it can be used when adenine is selected as the second purine.

  In the method according to the present embodiment, at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate based on the amount of change in the detected signal A known method may be used to calculate the amount. Specific examples include, but are not limited to, the following methods.

The amount of at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate in the sample may be calculated as follows. That is, in the enzyme cycling reaction according to the present embodiment, the reaction time is defined (for example, from the fifth minute to the seventh minute after the start of the reaction, etc.) By measuring the amount of change in the signal corresponding to the change, the amount of the test substance in the sample can be calculated. As a calibrator, for example, when calculating the amount of orthophosphoric acid, disodium hydrogen phosphate or dipotassium hydrogen phosphate solution, etc., when calculating ribose-1-phosphate, ribose-1-phosphate solution, Further, when calculating the amount of pyrophosphate, sodium pyrophosphate or potassium solution can be used. In addition, when calculating the amount of alkaline phosphatase activity, prior to the enzyme cycling reaction, the alkaline phosphatase (ALP) reaction is performed for a certain period of time, and after the reaction is stopped, the enzyme cycling reaction is performed, Using a known concentration of disodium hydrogen phosphate as a calibrator, the ALP activity in the sample can be calculated according to the following equation.
Enzyme activity (U / mL)
= (ΔAx / ΔAcal) × Cal (μmol / mL) × factor / reaction time (minutes)
ΔAx: Absorbance change amount when using sample ΔAcal: Absorbance change amount of calibrator Cal: Disodium hydrogen phosphate concentration in calibrator (μmol / mL)
Factor: Total reaction solution amount / sample addition amount ALP solution having a known activity can also be used for the calibrator.

Furthermore, this embodiment is a composition for measurement for measuring at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate. A thing,
(A) a purine nucleoside phosphorylase, a positive reaction for producing ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphate in the presence of a purine nucleoside having a first purine, and A purine nucleoside phosphorylase that catalyzes a reverse reaction and can use at least different purines in the forward reaction and the reverse reaction,
(B) a purine nucleoside having a first purine;
(C) a second pudding different from the first pudding;
A composition comprising:
A composition in contact with the sample,
When the measurement object is alkaline phosphatase, the sample is treated with a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphoric acid,
When the measurement target is pyrophosphoric acid, the sample is treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid.
It is a composition.

  The measurement object of the composition according to the present embodiment is at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate. Preferable measurement objects include alkaline phosphatase or pyrophosphate, and more preferably alkaline phosphatase. Orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate can be measured separately or simultaneously. In the case where orthophosphoric acid, ribose-1-phosphate and / or deoxyribose-1-phosphate are mixed in the sample, when it is desired to quantify only one or two kinds, a known method is required prior to the enzyme cycling reaction. The method may be used to perform a process such as converting a non-measurement object into another substance.

  The purine nucleoside phosphorylase in the composition according to the present embodiment is a positive enzyme that produces ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphate in the presence of a purine nucleoside having a first purine. The enzyme is not particularly limited as long as it is an enzyme that catalyzes the reaction and the reverse reaction. In the presence of a purine nucleoside having a first purine, whether or not the enzyme catalyzes a positive reaction for producing ribose-1-phosphate from orthophosphoric acid is confirmed by, for example, the above-described method for the method according to the present embodiment. Although it can determine by the method similar to description, it is not limited to this. Further, whether or not to catalyze the reverse reaction can be determined by, for example, the same method as described above for the method according to the present embodiment, but is not limited thereto.

  Although the purine nucleoside phosphorylase in the composition which concerns on this Embodiment is not specifically limited, For example, the purine nucleoside phosphorylase specified by EC2.4.2.1 is mentioned. Purine nucleoside phosphorylase in the composition according to the present embodiment widely exists from higher animals to microorganisms, and its origin is not particularly limited. For example, it can be obtained as a microorganism-derived enzyme from Bacillus stearothermophilus. In addition, microorganism-derived enzymes are commercially available from Toyobo Co., Ltd., and Bacillus-derived enzymes are commercially available from Asahi Kasei Pharma Corporation. Conventional methods may be used for culturing these derived cells or tissues and isolating and purifying the enzyme. For example, purine nucleoside phosphorylase whose purity has been improved to a practical level can be obtained by culturing microorganisms and combining known purification methods such as column chromatography. It can also be obtained by culturing and using transformed cells such as E. coli into which the gene has been incorporated. Furthermore, the purine nucleoside phosphorylase according to the composition of the present embodiment may be genetically modified for the purpose of improving enzyme properties. The genetically modified purine nucleoside phosphorylase can be obtained by, for example, isolating a gene encoding the enzyme protein by a conventional method and expressing the gene in a suitable host using a gene recombination technique.

  The “purine” in the composition according to the present embodiment means not only a naturally occurring purine but also a derivative or a similar substance synthesized chemically or enzymatically.

  Examples of the first purine in the composition according to the present embodiment include adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine, 7-methylguanine, 7-methyladenine, and 7-methylhypoxanthine. Examples include, but are not limited to, 2-amino-6-mercapto-7-methylpurine, 8-azahypoxanthine, 8-guanine, and derivatives thereof. Preferably, adenine, guanine, hypoxanthine, and xanthine are mentioned. More preferably, hypoxanthine and xanthine are mentioned.

  The “purine nucleoside” in the composition according to the present embodiment is a binding substance between purine and ribose, or a binding substance between purine and deoxyribose, and is not limited to a naturally occurring purine nucleoside, but a chemical or enzyme. It is meant to include synthetically derived derivatives thereof and similar substances. Examples of the purine nucleoside having the first purine in the composition according to the present embodiment include adenosine, guanosine, inosine, xanthosine deoxyadenine, deoxyguanine, deoxyinosine, and deoxyxanthosine. It is not limited to. Inosine and xanthosine are preferable.

  Examples of the purine nucleoside having the second purine include, but are not limited to, adenosine, guanosine, inosine, xanthosine, deoxyadenine, deoxyguanine, deoxyinosine, and deoxyxanthosine. Adenosine and guanosine are preferable.

  Examples of the second purine different from the first purine in the composition according to the present embodiment include adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, isoguanine, 7-methylguanine, 7 -Purine that was not selected as the first purine, such as -methyladenine, 7-methylhypoxanthine, 2-amino-6-mercapto-7-methylpurine, 8-azahypoxanthine, and 8-guanine However, it is not limited to these. Preferably, adenine, guanine, hypoxanthine, xanthine, etc. are mentioned, More preferably, adenine, guanine, etc. are mentioned.

  In the composition according to the present embodiment, the combination of the first purine and the second purine is not particularly limited. For example, a combination of any two of adenine, guanine, hypoxanthine, and xanthine may be used. Good. Preferably, a combination of hypoxanthine and guanine, a combination of hypoxanthine and adenine, a combination of xanthine and guanine, and a combination of xanthine and adenine.

  According to the composition according to the present embodiment, for example, the method according to the present embodiment can be performed, and the cycling reaction of the above formula (3) can be caused.

  In the composition according to the present embodiment, when the measurement target is alkaline phosphatase, a phosphate ester compound capable of hydrolyzing alkaline phosphatase is added to the sample, and the phosphate ester compound is hydrolyzed to obtain orthophosphoric acid. Used for samples after treatment. The phosphate compound is not particularly limited as long as it can hydrolyze alkaline phosphatase. For example, β-glycerophosphate, α-glycerophosphate, phenylphosphate, p-nitrophenylphosphate, phosphorylcholine, and NADP Etc. P-nitrophenyl phosphate is preferred. A known method may be used as the method for hydrolysis. For example, the phosphate ester compound as a substrate may be treated with alkaline phosphatase at a constant temperature, for example, 37 ° C. for a fixed time. Specifically, the enzyme treatment is performed in the presence of the phosphate ester compound using a buffer solution having a buffer region on the alkali side.

  In addition, when the measurement object is pyrophosphoric acid, the composition according to the present embodiment is used for a sample after subjecting pyrophosphoric acid to hydrolysis to obtain orthophosphoric acid. As the treatment for hydrolyzing pyrophosphoric acid to orthophosphoric acid, for example, a known chemical treatment using acid or alkali can be used, or enzymatic hydrolysis can be used. In the case of enzymatic treatment, for example, an enzyme reaction capable of converting known pyrophosphate into orthophosphate may be used. Specifically, pyrolysis of pyrophosphate by pyrophosphatase (EC 3.6.1.1), reverse reaction of pyruvate phosphate dikinase (EC 2.7.9.1), and the like can be used. Yeast-derived pyrophosphatase is commercially available.

  The sample to be examined with the composition according to the present embodiment is not particularly limited, but is, for example, a human or animal biological sample, preferably human blood, more preferably human serum. Whether or not the sample contains at least one of orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; May be unknown before using the composition, and after using the composition according to the present embodiment, the sample is at least one of orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate; It may be apparent that it does not contain phosphatase; or pyrophosphate.

  In the composition according to the present embodiment, the cycling reaction is not limited to this. For example, a known method may be referred to. Specifically, a method similar to the above description of the method according to the present embodiment can be employed, but is not limited thereto.

  According to the composition in the present embodiment, a signal corresponding to a change amount of at least one of a purine nucleoside having a first purine, a first purine, a second purine, and a purine nucleoside having a second purine. Can be detected. Preferably, the amount of change in the signal corresponding to the increased amount of the purine nucleoside having the first purine and / or the second purine can be detected, but is not limited thereto.

  As a method for detecting the amount of change, known HPLC methods and enzyme methods can be used, but are not limited thereto. When the HPLC method is used, a chelating agent or the like is added to the reaction solution to terminate the enzyme reaction, and then a reverse phase column or the like is appropriately selected for quantification. Information on these HPLC analysis methods is readily available from resin manufacturers.

  It can also be detected by enzymatic techniques. In this case, the composition according to the present embodiment further includes, for example, a detection enzyme that has substrate specificity for the first purine but no substrate property for the second purine, or the first purine. The purine nucleoside possessed does not have substrate specificity, but the second purine may contain a detection enzyme having substrate specificity. The detection enzyme is preferably an enzyme having substrate specificity for the first purine but not having substrate specificity for the second purine, but is not limited thereto. As an enzymatic method, for example, the first purine has substrate specificity, but the second purine has no substrate property, or the purine nucleoside having the first purine has substrate specificity. A detection enzyme having substrate specificity is used for a purine nucleoside having no second but having a second purine, and a signal corresponding to an increase in the amount of the purine nucleoside having the first purine and / or the second purine. There is a method for detecting the amount of change. The first purine has substrate specificity, but the second purine does not have substrate specificity, and detects the amount of change in the signal corresponding to the increased amount of the first purine. A method is mentioned as a preferable method.

  Examples of the detection enzyme of the composition according to the present embodiment include xanthine dehydrogenase (EC 1.2.1.37) having substrate specificity for hypoxanthine and / or xanthine, and substrate specificity for hypoxanthine and / or xanthine. Xanthine oxidase (EC 1.17.3.2), adenine deaminase with substrate specificity for adenine (EC 3.5.4.4), and guanine deaminase with substrate specificity for guanine (EC 3.5) 4.3) and the like, but are not limited thereto. When xanthine dehydrogenase (EC 1.2.1.37) is used as a detection enzyme, NAD or thioNAD may further be contained in the composition according to the present embodiment. When carrying out these methods specifically, for example, when inosine or xanthosine is selected as the purine nucleoside having the first purine and adenine or guanine is selected as the second purine, the amount of hypoxanthine and / or xanthine increased In order to detect the amount of change in the signal corresponding to the above, if xanthine dehydrogenase (Asahi Kasei Pharma Co., Ltd., T134) is used, adenine or guanine is not detected, but hypoxanthine which is a positive reaction change product of inosine or xanthosine or Only xanthine can be detected. Similarly, since xanthine oxidase commercially available from Toyobo Co., Ltd. is said not to act on adenine, it can also be used when adenine is selected as the second purine.

  These detection enzymes are commercially available as described above and can be easily obtained. In addition, since adenine deaminase and guanine deaminase are known to exist in bacteria and the like, they can be easily obtained by culturing and purifying according to conventional methods with reference to the literature on these. Furthermore, when the gene sequences of the enzymes that produce these are known, the genes can be obtained from cDNA or synthesis and produced as recombinants. For example, a paper on the expression of guanine deaminase derived from E. coli (J. Bacteriol 182, 4658-4660, 2000) has been reported.

  If the composition according to the present embodiment is used, at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or The amount of pyrophosphate can be calculated. For example, a known method as described above may be used as the calculation method. Specifically, it can be determined by the same method as described above for the method according to the present embodiment, but is not limited thereto.

  The composition according to the present embodiment may be a reagent kit. The “reagent kit” in the present embodiment may be divided into a plurality of reagents, but may be combined into one. For example, the composition according to the present embodiment may be appropriately distributed into two or more reagent kits. For example, in the case of a composition for quantifying at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate, inosine, guanine, and NAD are used as one reagent, xanthine dehydrogenase, and Purine nucleoside phosphorylase can be distributed to the other reagent. Any of these components can also be included in a separate reagent kit.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.

Example 1 Determination of inorganic phosphorus by purine nucleoside phosphorylase (PNPL) in the presence of inosine and guanine (reaction solution)
50 mmol / L Tris-HCl (pH 8.0)
2.5mmol / L NAD
2 mmol / L inosine 0.5 mmol / L guanine 1u / mL or 5u / mL PNPL (PNPLII, manufactured by Asahi Kasei Pharma)
1u / mL xanthine dehydrogenase (XDHII, manufactured by Asahi Kasei Pharma)

  0.01 mL of a 0, 0.1, 0.2, 0.4, 1 mmol / L dipotassium hydrogen phosphate aqueous solution was added to 1 mL of the reaction solution, and reacted at 37 ° C. After the addition of the dipotassium hydrogen phosphate aqueous solution, the change in absorbance at 340 nm from the third minute to the fourth was measured. In each case of PNPL of 1 u / mL and 5 u / mL, the reaction solution to which the dipotassium hydrogen phosphate aqueous solution was not added was used as a reagent blank and subtracted from each absorbance change amount. The results are shown in FIG. 1. From FIG. 1, as the amount of purine nucleoside phosphorylase increases, the product of cycling reaction increases, the amount of hypoxanthine that is a substrate of xanthine dehydrogenase increases, and as the amount of dipotassium hydrogen phosphate increases, hypoxanthine that is a cycling reaction product. The amount of was shown to increase.

Example 2 Determination of inorganic phosphorus with purine nucleoside phosphorylase (PNPL) in the presence of inosine and adenine (Reaction solution)
50 mmol / L Tris-HCl (pH 8.0)
2.5mmol / L NAD
1 mmol / L inosine 0.5 mmol / L adenine 1 u / mL PNPL (PNPLII, manufactured by Asahi Kasei Pharma)
1u / mL xanthine dehydrogenase (XDHII, manufactured by Asahi Kasei Pharma)

  0.05 mL of 0, 10, 20, 30, 40, 50 umol / L dipotassium phosphate aqueous solution was added to 1 mL of the reaction solution, and the reaction was allowed to proceed at 37 ° C. After the addition of the dipotassium hydrogen phosphate aqueous solution, the change in absorbance at 340 nm from the third minute to the fourth was measured. The reaction liquid to which the dipotassium hydrogen phosphate aqueous solution was not added was used as a reagent blank, and the absorbance change amount of the reagent blank was subtracted from each absorbance change amount. The results are shown in FIG. FIG. 2 shows that the amount of hypoxanthine increases as the amount of dipotassium hydrogen phosphate increases.

Example 3 Comparison of Purine Nucleoside Phosphorylases (PNPL) with Different Origins (Reaction Solution)
50 mmol / L Tris-HCl (pH 8.0)
2.5mmol / L NAD
1mmol / L inosine 0.5mmol / L adenine 5u / mL PNPL (PNP-301, Toyobo)
1u / mL xanthine dehydrogenase (XDHII, manufactured by Asahi Kasei Pharma)

  Toyobo PNP-301 was obtained as a purine nucleoside phosphorylase having a different origin from that of Example 2. According to the catalog, the relative activity to guanosine is 60% when inosine is 100%. When 5 u / mL was added to the reaction solution with the indicated activity, almost the same signal as in Example 2 was obtained.

Example 4 Determination of inorganic phosphorus with purine nucleoside phosphorylase (PNPL) in the presence of inosine and adenine (detection reaction with xanthine oxidase)
(Reaction solution)
50 mmol / L Tris-HCl (pH 8.0)
2 mmol / L inosine 3 mmol / L 4-aminoantipyrine 1.5 mmol / L N, N-bis (4-sulfobutyl) -3-methylaniline disodium (N, N-Bis (4-sulfobutyl) -3-methylalanine, disodium salt, TODB, manufactured by Dojin Chemical)
5 u / mL peroxidase (Sigma)
1u / mL PNPL (PNPLII, manufactured by Asahi Kasei Pharma)
1 u / mL xanthine oxidase (XOD: manufactured by Toyobo)

  0.05 mL of 0, 40, 80, 120, 160, 200 umol / L dipotassium hydrogen phosphate aqueous solution was added to 1 mL of the reaction solution, and pre-warmed at 37 ° C. for 3 minutes. Subsequently, adenine was added in the reaction solution so as to be 0.5 mmol / L, and the reaction was started. From the xanthine produced by the PNPL reaction, hydrogen peroxide is produced by xanthine oxidase, and in the presence of peroxidase, N-bis (4-sulfobutyl) -3-methylaniline disodium and 4-aminoantipyrine are oxidized. Condensation to produce a red quinone dye and the absorbance at 555 nm was monitored. After the addition of adenine, the change in absorbance from the 3rd minute to the 5th minute was measured. The reaction liquid to which the dipotassium hydrogen phosphate aqueous solution was not added was used as a reagent blank, and the absorbance change amount of the reagent blank was subtracted from each absorbance change amount. The results are shown in FIG. From FIG. 3, it was shown that the change amount of the product of the cycling reaction can be detected also by the detection reaction with xanthine oxidase.

Example 5 Purine nucleoside phosphorylase (PNPL) reaction: Comparison between a combination of inosine and guanine and a combination of deoxyinosine and guanine (reaction solution)
50 mmol / L Tris-HCl (pH 8.0)
2.5mmol / L NAD
2mmol / L inosine or deoxyinosine 5u / mL PNPL (Toyobo)
1u / mL xanthine dehydrogenase (XDHII, manufactured by Asahi Kasei Pharma)

  0.05 mL of 0, 10, 20, 30, 40 umol / L dipotassium hydrogen phosphate aqueous solution was added to 1 mL of the reaction solution, and pre-warmed at 37 ° C for 3 minutes. Subsequently, guanine was added to the reaction liquid so that it might become 0.5 mmol / L, and reaction was started. Hypoxanthine produced by the PNPL reaction was combined with xanthine dehydrogenase, and the change in absorbance at 340 nm as the reduced NAD increased was monitored. Here, after the addition of guanine, the change in absorbance from the 3rd minute to the 5th minute was measured. The reaction liquid to which the dipotassium hydrogen phosphate aqueous solution was not added was used as a reagent blank, and the absorbance change amount of the reagent blank was subtracted from each absorbance change amount. The same operation was performed on the inosine changed to deoxyinosine. The results are shown in FIG. The combination of inosine and guanine and the combination of deoxyinosine and guanine showed almost the same reactivity.

Example 6 Measurement of Alkaline Phosphatase (ALP) Activity (Reaction Solution According to Example 6)
(R-1)
1 mol / L EAE hydrochloric acid buffer (pH 9.9)
15 mmol / L p-nitrophenyl phosphate 0.5 mmol / L MgCl ₂
2 mmol / L inosine (R-2)
1 mol / L PIPES-NaOH (pH 6.2)
10mmol / L EDTA
10u / mL PNPL (Asahi Kasei Pharma Corporation)
0.5 mmol / L guanine 2.5 u / mL xanthine dehydrogenase (XDHII, manufactured by Asahi Kasei Pharma)
2mmol / L NAD

(Comparative control method)
(R-1)
Same as above (R-2)
1 mol / L PIPES-NaOH (pH 6.2)
10mmol / L EDTA
1u / mL PNPL
2.5u / mL xanthine dehydrogenase (XDHII, manufactured by Asahi Kasei Pharma)
2mmol / L NAD

  A 0.5 u / mL alkaline phosphatase (ALP, derived from human liver, manufactured by Asahi Kasei Pharma) solution (20 mmol / L Tris-HCl (pH 8.5), 0.01% BSA) was prepared. First, activity was measured as follows, and it was confirmed that there was activity as indicated.

  After pre-warming 0.5 mL of R-1 at 37 ° C., 0.01 mL of ALP enzyme solution was added and allowed to react accurately for 5 minutes. Subsequently, 0.5 mL of R-2 of the comparative control method was added, and inorganic phosphorus released by the ALP reaction was quantified at 340 nm as the amount of reduced NAD. An absorbance of 0.304 was obtained with respect to a theoretical absorbance of 0.315.

  Next, the ALP enzyme solution was further diluted 1000, 2000, and 5000 times with the same diluent. Each of these three types of enzyme solutions was similarly reacted with R-1 at 37 ° C. for 5 minutes. Thereafter, 0.5 mL of R-2 according to Example 6 was added, the absorbance at 340 nm was monitored, and the amount of change in absorbance from the 3rd minute to the 8th minute after the addition of R-2 was measured. What used the physiological saline instead of the enzyme was made into the reagent blank, it subtracted from each measured value, and it plotted with respect to the enzyme concentration (X-axis). In addition, the theoretical absorbance measured by the comparative method, which is a conventional method, was also plotted. The results are shown in FIG.

  The amount of change in absorbance for 5 minutes according to Example 6 was about 250 times the theoretical absorbance according to the conventional method. This indicates that the cycling reaction according to Example 6 for 5 minutes enhanced the signal 250 times that of the conventional method. In Example 6, quantification of ALP activity of at least 100 mU / L was possible.

Example 7 Measurement of pyrophosphate (reaction solution)
100 mmol / L Tris-HCl (pH 7.5)
1 mmol / L adenosine monophosphate (AMP)
0.5 mmol / L phosphoenolpyruvate (PEP)
5u / mL pyruvate orthophosphate dikinase (PPDK)
1 mmol / L inosine 1 u / mL xanthine dehydrogenase (XDHII, manufactured by Asahi Kasei Pharma)
2.5mmol / L NAD

As an enzyme for converting pyrophosphate into inorganic phosphorus, pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) (Patent No. 5352788) derived from Thermotoga Maritima DSM 3109 strain was used. PPDK acts on adenosine 5 ′ monophosphate, phosphoenolpyruvate and pyrophosphate to catalyze reactions that produce adenosine 5 ′ triphosphate, pyruvate, and phosphate and vice versa. The enzyme action of pyruvate orthophosphate dikinase is as shown in the following formula in the presence of magnesium ion or the like (see Enzyme Handbook, Asakura Shoten, 1984).
ATP + pyruvic acid + orthophosphoric acid <=> AMP + PEP + pyrophosphoric acid

  0.05 mL of 0, 0.5, 1, 2, 4, 10 umol / L sodium pyrophosphate aqueous solution was added to 1 mL of the reaction solution, and prewarmed at 37 ° C. for 3 minutes. Subsequently, guanine was added to the reaction liquid so that it might become 0.5 mmol / L, and reaction was started. After the addition of guanine, the amount of change in absorbance was measured from the 3rd minute to the 5th minute. The reaction solution to which the pyrophosphoric acid aqueous solution was not added was used as a reagent blank, and the absorbance change amount of the reagent blank was subtracted from each absorbance change amount. The results are shown in FIG.

Example 8 Determination of inorganic phosphorus with purine nucleoside phosphorylase (PNPL) in the presence of guanosine and hypoxanthine (detection reaction with guanine deaminase)
(Reaction solution)
50 mmol / L Tris-HCl (pH 7.5)
2 mmol / L hypoxanthine 1 mmol / L ATP
0.5mmol / L Deamide NAD (Asahi Kasei Pharma)
2 mmol / L MgCl ₂
1 mmol / L sodium cholate 2u / mL purine nucleoside phosphorylase 2u / mL NAD synthetase (NADSII, manufactured by Asahi Kasei Pharma)
2u / mL 12α-hydroxysteroid dehydrogenase (12α-HSDII, manufactured by Asahi Kasei Pharma)
2u / mL guanine deaminase (from rabbit liver, manufactured by Wako Pure Chemical Industries)

  Purine nucleoside phosphorylase enzyme cycling reaction was performed in the presence of guanosine and hypoxanthine. The produced guanine was converted into xanthine and ammonia by guanine deaminase, and the rate of increase in absorbance at 340 nm was measured as reduced NAD in the presence of NADSII and 12α-HSDII. First, 0.02 m of 0, 20, 40, 60, 80, 100 mol / L dipotassium hydrogenphosphate aqueous solution was added to 1 mL of the above reaction solution, and after preheating, 0.1 mL of 20 mmol / L guanosine aqueous solution was added. The reaction was started at 37 degrees. After the addition of guanosine, the change in absorbance at 340 nm was measured from the third minute to the fourth. A reagent blank that was not added with a dipotassium hydrogen phosphate aqueous solution was used as a reagent blank and subtracted from each amount of change in absorbance. The results are shown in FIG.

Claims (41)

At least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate,
(1) Purine nucleoside phosphorylase, which catalyzes a forward reaction for producing ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphoric acid in the presence of purine nucleoside, and a reverse reaction thereof. A sample of a purine nucleoside phosphorylase that can utilize at least different purines in the forward reaction and the reverse reaction, a purine nucleoside having a first purine, and a second purine that is different from the first purine. A step of bringing into contact with and causing a cycling reaction of the following formula (1):
(2) A change amount of a signal corresponding to a change amount of at least one of the purine nucleoside having the first purine, the first purine, the second purine, and the purine nucleoside having the second purine. Detecting step;
(3) Based on the detected amount of change in signal, calculate the amount of at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate And a process of
Including
When the measurement object is alkaline phosphatase, the condition in which the phosphate ester compound can be hydrolyzed to produce phosphate in the presence of the phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphate The sample has been processed at
When the measurement target is pyrophosphoric acid, the sample is treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid.
Measuring method.   The method according to claim 1, wherein the measurement target is at least one of orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate. The measurement object is alkaline phosphatase,
The sample is treated in the presence of a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphoric acid under conditions that allow the phosphate ester compound to be hydrolyzed to produce phosphate. The method according to claim 1, wherein the amount of alkaline phosphatase is calculated based on the amount of change in the detected signal. The measurement object is pyrophosphoric acid, and the sample is treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid,
Based on the amount of change in the detected signal, the amount of pyrophosphate is calculated.
The method of claim 1.   The step (2) is a step of detecting a change amount of a signal corresponding to an increase amount of a purine nucleoside having the first purine and / or the second purine. The method according to any one of the above.   In the step (2), the first purine has a substrate specificity but the second purine has no substrate specificity, or the purine nucleoside having the first purine has a substrate specificity. Does not have substrate specificity, but for the purine nucleoside having the second purine, a purine having the first purine and / or the second purine is used by using a detection enzyme having substrate specificity. The method according to any one of claims 1 to 5, wherein an amount of change in signal corresponding to an increased amount of nucleoside is detected.   In the step (2), the first purine has a substrate specificity, but the second purine has no substrate specificity, and the detection amount of the first purine is increased. The method according to any one of claims 1 to 6, wherein the amount of change in the signal corresponding to is detected.   The combination of the first purine and the second purine different from the first purine is a combination of hypoxanthine and guanine, a combination of hypoxanthine and adenine, a combination of xanthine and guanine, or xanthine The method according to any one of claims 1 to 7, which is a combination of adenine and adenine.   The method according to any one of claims 1 to 8, wherein the first purine is hypoxanthine or xanthine.   9. The method according to any one of claims 1 to 8, wherein the first purine is guanine.   9. The method according to any one of claims 1 to 8, wherein the first purine is adenine.   In the above step (2), using xanthine dehydrogenase having substrate specificity for hypoxanthine and / or xanthine, in the presence of thio-NAD or NAD, the amount of change in signal corresponding to the amount of hypoxanthine and / or xanthine increased. The method according to claim 9, wherein the method is detected.   10. In the step (2), the amount of change in signal corresponding to the amount of hypoxanthine and / or xanthine increased is detected using xanthine oxidase having substrate specificity for hypoxanthine and / or xanthine. the method of.   The method according to claim 10, wherein in the step (2), a change in signal corresponding to an increased amount of guanine is detected using guanine deaminase having substrate specificity for guanine.   The method according to claim 11, wherein in the step (2), an amount of change in signal corresponding to an increased amount of adenine is detected using adenine deaminase having substrate specificity for adenine.   The purine nucleoside having the first purine is inosine or deoxyinosine;
  The first purine is hypoxanthine;
  In the step (2), the amount of change in the signal corresponding to the amount of change in the hypoxanthine is detected using xanthine dehydrogenase or xanthine oxidase having substrate specificity for the hypoxanthine,
  The method of claim 1.   The method of claim 16, wherein the second purine is guanine or adenine.   The purine nucleoside having the first purine is xanthosine or deoxyxanthosine,
  The first purine is xanthine;
  In the step (2), the amount of change in signal corresponding to the amount of change in xanthine is detected using xanthine dehydrogenase or xanthine oxidase having substrate specificity for the xanthine,
  The method of claim 1.   The method of claim 18, wherein the second purine is guanine or adenine. A method for measuring alkaline phosphatase, comprising:
(1) A purine nucleoside phosphorylase (EC 2.4.2.1), which generates ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphoric acid in the presence of inosine, And purine nucleoside phosphorylase that can catalyze the reverse reaction and can use guanine in the reverse reaction, inosine, and guanine are brought into contact with a sample, and a cycling reaction of the following formula (2) is caused to react:
(2) using xanthine dehydronase, detecting a change amount of a signal corresponding to an increased amount of hypoxanthine;
(3) calculating the amount of alkaline phosphatase based on the amount of change in the detected signal;
Including
In the presence of a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphate, the sample is treated under conditions that allow the phosphate ester compound to be hydrolyzed to produce phosphate. ,
13. A method according to any one of claims 1, 3, 5 to 9, and 12. A measurement composition for measuring at least one of orthophosphate, ribose-1-phosphate, and deoxyribose-1-phosphate; alkaline phosphatase; or pyrophosphate,
(A) a purine nucleoside phosphorylase, a positive reaction for producing ribose-1-phosphate and / or deoxyribose-1-phosphate from orthophosphate in the presence of a purine nucleoside having a first purine, and A purine nucleoside phosphorylase that catalyzes a reverse reaction and can use at least different purines in the forward reaction and the reverse reaction,
(B) a purine nucleoside having a first purine;
(C) a second pudding different from the first pudding;
A composition comprising:
A composition in contact with the sample,
When the measurement object is alkaline phosphatase, the condition in which the phosphate ester compound can be hydrolyzed to produce phosphate in the presence of the phosphate ester compound that can be hydrolyzed with alkaline phosphatase to produce orthophosphate The sample has been processed at
When the measurement target is pyrophosphoric acid, the sample is treated under conditions that allow pyrophosphoric acid to be hydrolyzed to produce orthophosphoric acid.
Composition. The composition according to claim 21 , wherein the measurement object is orthophosphoric acid, ribose-1-phosphate, and / or deoxyribose-1-phosphate. The measurement object is alkaline phosphatase,
Contact with the sample treated in the presence of a phosphate ester compound that can be hydrolyzed with alkaline phosphatase to generate orthophosphate in the presence of the phosphate ester compound being hydrolyzed to produce phosphate. The composition of claim 21 , wherein The composition according to claim 21 , wherein the measurement target is pyrophosphate, and the sample is contacted with the sample that has been treated under conditions that allow pyrophosphate to be hydrolyzed to produce orthophosphoric acid. The purine nucleoside phosphorylase is EC 2.4.2.1, and ribose-1-phosphate and / or deoxyribose-1-phosphate is converted from orthophosphate in the presence of the purine nucleoside having the first purine. 25. The composition according to any one of claims 21 to 24 , which catalyzes a normal reaction to be generated and a reverse reaction thereof, and at least different purines can be used in the normal reaction and the reverse reaction, respectively. The first purine has substrate specificity, but the second purine has no substrate specificity, or the purine nucleoside having the first purine has substrate specificity. 26. The composition according to any one of claims 21 to 25 , wherein the purine nucleoside having the second purine further comprises a detection enzyme having substrate specificity. 27. The composition according to any one of claims 21 to 26 , wherein the first purine contains a detection enzyme that has substrate specificity, but the second purine does not have substrate specificity. The combination of the first purine and the second purine different from the first purine is a combination of hypoxanthine and guanine, a combination of hypoxanthine and adenine, a combination of xanthine and guanine, or xanthine 28. A composition according to any one of claims 21 to 27 , which is a combination of and adenine. 29. A composition according to any one of claims 21 to 28 , wherein the first purine is hypoxanthine or xanthine. 29. A composition according to any one of claims 21 to 28 , wherein the first purine is guanine. 29. A composition according to any one of claims 21 to 28 , wherein the first purine is adenine. Xanthine dehydrogenase having substrate specificity for hypoxanthine and / or xanthine;
NAD or thio-NAD,
30. The composition of claim 29 , comprising: 30. The composition of claim 29 , comprising hypoxanthine and / or xanthine oxidase having substrate specificity for xanthine. 32. The composition of claim 30 , comprising guanine deaminase having substrate specificity for guanine. 32. The composition of claim 31 , comprising adenine deaminase having substrate specificity for adenine.   The purine nucleoside having the first purine is inosine or deoxyinosine;
  The first purine is hypoxanthine;
  Further comprising xanthine dehydrogenase or xanthine oxidase having substrate specificity for the hypoxanthine as a detection enzyme,
  The composition according to claim 21.   37. The composition of claim 36, wherein the second purine is guanine or adenine.   The purine nucleoside having the first purine is xanthosine or deoxyxanthosine,
  The first purine is xanthine;
  It further contains xanthine dehydrogenase or xanthine oxidase having substrate specificity for the xanthine as an enzyme for detection.
  The composition according to claim 21.   40. The composition of claim 38, wherein the second purine is guanine or adenine. A measurement composition for measuring alkaline phosphatase,
(A) a purine nucleoside phosphorylase, which catalyzes a forward reaction for producing ribose-1-phosphate from orthophosphoric acid in the presence of inosine, and its reverse reaction, and can use guanine in the reverse reaction;
(B) inosine;
(C) guanine,
(D) xanthine dehydrogenase;
(E) NAD or thio DNA;
A composition comprising:
Used for samples treated with phosphate ester compounds that can be hydrolyzed with alkaline phosphatase to produce orthophosphoric acid,
The composition according to any one of claims 21 , 23 , 25 to 29 , and 32 . 41. At least one of orthophosphoric acid, ribose-1-phosphate, and deoxyribose-1-phosphate comprising the composition of any one of claims 21 to 40 ; alkaline phosphatase; or for measuring pyrophosphate kit.