JP4045315B2 - Reagent for measuring calcium in sample and measuring method - Google Patents

Description

The present invention relates to a calcium measuring reagent and a measuring method for measuring calcium in a sample.
With the reagent and method for measuring calcium in a sample according to the present invention, it is possible to measure calcium in a sample at a low cost, further increase the measurement sensitivity, and accurately measure the calcium concentration with good reproducibility. It is something that can be done.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

  About 1,200 g of calcium is present in the human body, about 99% of which is present in bones and teeth, and the remaining about 1% is present in body fluids such as blood and muscles. This 1% calcium plays an important role in life support and activities, such as stopping bleeding, working nerves, and exercising muscles. The calcium concentration in blood varies depending on calcium absorption abnormality, bone disease, endocrine disease, hypertension, arteriosclerosis, and the like, and its measurement is extremely important clinically.

  At present, methods used for measuring calcium include an atomic absorption method, an electrode method, and an o-cresolphthalein complexone method (hereinafter sometimes abbreviated as OCPC method). These methods may require expensive equipment or require sample pretreatment. In particular, the OCPC method has drawbacks such as being affected by magnesium ions and the absorbance changing depending on temperature and measurement time.

For this reason, recently, as a measurement method using activation or inhibition of an enzyme by calcium ions, measurement using activation of phospholipase D by calcium ions (see, for example, Patent Documents 1 and 2), pyrubin by calcium ions. Measurements using inhibition of acid kinase activity (for example, see Patent Document 3), measurements using activation of α-amylase by calcium ions (for example, see Patent Document 4), and the like have been proposed.
Japanese Patent Laid-Open No. 62-195297 Japanese Patent Laid-Open No. 4-187098 JP-A-2-142498 JP-A-2-276597

However, these methods for measuring calcium in samples (measurement reagents) have the disadvantage that the measurement range is limited if they use inhibition by calcium ions. Moreover, the thing using activation of (alpha) -amylase had the problem that it was easy to receive to the influence of (alpha) -amylase in a sample. Moreover, although the thing using the activation by the calcium ion of phospholipase D is excellent in the specificity and the measurement sensitivity, the product produced | generated by reaction with the substrate of phospholipase D and the phospholipase D is intervened by coupling enzyme. In order to perform the measurement, complicated operations such as pH adjustment were required.
And these calcium measuring reagents and measuring methods have high measuring costs.

  Therefore, an object of the present invention is to enable measurement of the calcium concentration in a sample at a low cost while suppressing the measurement cost in a measurement system using activation of phospholipase C and / or phospholipase D by calcium ions. It is an object to provide a measuring reagent and a measuring method capable of increasing the sensitivity and accurately measuring the calcium concentration with good reproducibility.

As a result of intensive studies aimed at solving the above-mentioned problems, the present inventor, as a substrate for phospholipase C and / or phospholipase D, in the measurement utilizing the activation of phospholipase C and / or phospholipase D by calcium ions. Represented by the following general formula (I) (wherein X1 is a chromophoric group having a nitrophenyl group , X2 is a chromophoric group having a hydrogen atom or a nitrophenyl group, and X3 is a hydrogen atom). It was found that the calcium in the sample can be accurately measured by using the obtained compound. Furthermore, the measurement range of calcium concentration in the sample can be expanded by allowing the measurement system to coexist with at least one substance selected from the group consisting of metal ions, chelating agents, amines, basic amino acids, or heterocyclic compounds. The headline and the present invention were completed.

That is, the present invention provides the following inventions.
(1) at least phospholipase C and / or phospholipase D, in the general formula (I) (the following formula as a substrate for phospholipase C and / or phospholipase D, X1 is a chromogenic group having Nitorofu Eniru group, X2 is a chromogenic group having a hydrogen atom or a nitrophenyl group, X3 contains a compound represented by a hydrogen atom), calcium measuring reagent in the sample.

(2) The reagent for measuring calcium in a sample according to (1) above , which comprises a substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to 0.03 to 10 mM.

(3) The method according to (1) or (2) above, comprising at least one substance selected from the group consisting of metal ions, chelating agents, amines, basic amino acids, or heterocyclic compounds Reagent for measuring calcium in a sample.

(4) The reagent for measuring calcium in a sample according to any one of (1) to (3) above, which comprises a nonionic surfactant and / or an anionic surfactant.
(5) chromogenic group having a nitro phenyl group, a 4-nitrophenyl group, 2-chloro-4 - characterized in that it is a nitrophenyl group, or 2,6-dichloro-4-nitrophenyl group, wherein The reagent for measuring calcium in a sample according to any one of (1) to (4).

( 6 ) A method for measuring calcium in a sample,
a) The following general formula (I) as a substrate of the sample, phospholipase C and / or phospholipase D, and phospholipase C and / or phospholipase D (wherein X1 is a chromophoric group having a nitrophenyl group ; X2 is a chromophoric group having a hydrogen atom or a nitrophenyl group, and X3 is a hydrogen atom), and reacting to release a chromophoric group having a nitrophenyl group from the compound. ,as well as

b) A method for measuring calcium in a sample, comprising a step of colorimetrically measuring the chromophoric group having a liberated nitrophenyl group .

( 7 ) In the sample according to ( 6 ), in the step a), a substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to calcium is adjusted to 0.03 to 10 mM. Calcium measurement method.

( 8 ) In the step (a), at least one substance selected from the group consisting of a metal ion, a chelating agent, or a nitrogen-containing compound is allowed to coexist, ( 6 ) or ( 7 ) Method for measuring calcium in a sample.

( 9 ) The sample according to any one of ( 6 ) to ( 8 ) above, wherein in the step a), a nonionic surfactant and / or an anionic surfactant is contained. Calcium measurement method.
(10) chromogenic group having a nitro phenyl group, a 4-nitrophenyl group, 2-chloro - characterized in that it is a 4-nitrophenyl group, or 2,6-dichloro-4-nitrophenyl group, wherein The method for measuring calcium in a sample according to any one of (6) to (9).

  In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, by using the compound represented by the above general formula (I) as a substrate of phospholipase C and / or phospholipase D, no other enzyme is used. Both can now be measured, and calcium in the sample can be measured inexpensively and accurately.

Furthermore, the calcium concentration in the sample is obtained by containing or coexisting at least one substance selected from the group consisting of metal ions, chelating agents, amines, basic amino acids, or heterocyclic compounds during the measurement reagent or measurement reaction. The measurement range can be expanded to a high concentration range.

  Moreover, the progress (time course) of the reaction at the time of measuring the calcium concentration in the sample can be stabilized by containing or coexisting a nonionic surfactant during the measurement reagent or measurement reaction.

  Then, by including or coexisting an anionic surfactant during the measurement reagent or measurement reaction, the sensitivity of the calcium concentration measurement is increased, and even a trace amount of calcium can be measured.

  In addition, by containing or coexisting an anionic surfactant during the measurement reagent or measurement reaction, even if phosphodiesterase is mixed in the sample, The reaction of liberating the chromophoric group of the substrate composed of the compound represented by the formula (I) can be suppressed, and the occurrence of a positive error in the measured value can be prevented.

(1) Phospholipase C and / or phospholipase D substrate In the present invention, the following general formula (I) (wherein X1 is a chromophoric group having a nitrophenyl group, and X2 is a hydrogen atom or a nitrophenyl group). And a compound represented by X3 is a hydrogen atom) is used as a substrate for phospholipase C and / or phospholipase D.

In the compound represented by the general formula (I), the chromophoric group having a nitrophenyl group includes an absorbance or transmission in an ester bond to a phosphate group and in a state liberated from the phosphate group by hydrolysis. Any chromophoric group having a nitrophenyl group having a difference in signal such as rate may be used.

The chromophoric group having a nitrophenyl group is preferably a group having a large difference in signal such as absorbance or transmittance between an ester bond to a phosphate group and a state free from the phosphate group.

Then, the chromogenic group X1 having a nitrophenyl group, the chromogenic group X2 having a nitro phenyl group, each may be the same or may be different.
A chromogenic group X1 having this nitrophenyl group, a chromogenic group X2 having a nitro phenyl group, if different from, each measurement wavelength range, ie, whether or near maximum absorption wavelength is the same Some are preferred.

The chromogenic group having a nitro phenyl group in the compound represented by the general formula (I), for example, 4-nitrophenyl group, 2-chloro-4-nitrophenyl group, or 2,6-dichloro-4 -A nitrophenyl group etc. can be mentioned.

Further, among the compounds represented by the general formula (I), as a compound in the case where X2 is not a chromophoric group having a nitrophenyl group , for example, 4-nitrophenyl phosphate [hereinafter referred to as 4-NPP] 4-nitrophenyl phenyl phosphate (hereinafter sometimes referred to as 4-NPPP), 2-chloro-4-nitrophenyl phosphate (hereinafter sometimes referred to as 4-CNPP), 2-chloro-4 -Nitrophenyl phenyl phosphate (hereinafter sometimes referred to as 4-CNPPP), 2,6-dichloro-4-nitrophenyl phosphate (hereinafter also referred to as 2,6-DCNPP), or 2,6-dichloro 4-nitro phenyl phosphate [hereinafter sometimes referred to as 2,6-DCNPPP], or may be their salts, and the like.

Furthermore, among the compounds represented by the above general formula (I), examples of the compound in which X2 is a chromophoric group having a nitrophenyl group include bis (4-nitrophenyl) phosphate [hereinafter referred to as Bis-4]. -NPP], bis (2-chloro-4-nitrophenyl) phosphoric acid (hereinafter sometimes referred to as Bis-4-CNPP), or bis (2,6-dichloro-4-nitrophenyl) phosphorus acid [hereinafter sometimes referred to as Bis-2,6-DCNPP], or may be their salts, and the like.

Examples of the compound represented by the general formula (I) in the present invention include bis (4-nitrophenyl) phosphoric acid, bis (2-chloro-4-nitrophenyl) phosphoric acid, or bis (2,6-dichloro). 4-nitrophenyl) phosphoric acid, or preferably ones even X2 in addition to X1 such as these salts are chromogenic group having a nitro Rofeniru group.
It if chromogenic group having a nitro phenyl group liberated from the phosphate groups by the catalytic action of phospholipase C and / or phospholipase D is activated by calcium, X2 is not a chromogenic group having nitro Rofeniru group X1 In contrast, when X1 and X2 are chromophoric groups having a nitrophenyl group , both X1 and X2 are liberated from the phosphate group, and the resulting signal is doubled. This is because the sensitivity can be measured.

In addition, it is preferable that the density | concentration at the time of making the compound represented with the said general formula (I) mix and react with a sample and phospholipase C and / or phospholipase D exists in the range of 0.02-200 mM. A range of 1 to 100 mM is particularly preferable.
The concentration of the compound represented by the general formula (I) contained in the calcium measurement reagent in the sample of the present invention is 0. The concentration when the sample and the phospholipase C and / or phospholipase D are mixed and reacted. What is necessary is just to set so that it may become 0.1-100 mM, so that it may become 02-200 mM.
For example, what is necessary is just to contain the compound represented by the said general formula (I) so that it may become 0.02-200 mM, Preferably it will be 0.1-100 mM.

  In addition, although the compound represented with the said general formula (I) can be easily obtained as a commercial item, it can also be easily manufactured by combining a synthetic method well-known to those skilled in the art suitably.

(2) Phospholipase C
In the present invention, phospholipase C (EC 3.1.4.3) is an enzyme that hydrolyzes an ester bond between a diacylglycero group and a phosphocholine group of glycerophospholipid to release diacylglycerol and phosphocholine.

As the phospholipase C used in the present invention, any phospholipase C can be used, for example, derived from microorganisms such as Streptomyces, Clostridium, Bacillus or Pseudomonas, or higher animals brain, liver, spleen or Examples include those derived from animal tissues such as erythrocytes.
Examples of the phospholipase C include those prepared by gene recombination techniques in which genes such as microorganisms are incorporated into microorganisms such as Escherichia coli, or phospholipases C prepared from microorganisms whose properties have been improved by gene modification or the like.

The activity value when this phospholipase C is mixed and reacted with the sample and the compound represented by the general formula (I) is preferably in the range of 0.01 to 10 units / mL, A range of 5 units / mL is particularly preferred.
The activity value of phospholipase C contained in the calcium measurement reagent in the sample of the present invention is 0.01 to 10 when the sample and the compound represented by the general formula (I) are mixed and reacted. What is necessary is just to set so that it may become 0.01-5 units / mL so that it may become a unit / mL.
For example, phospholipase C may be contained so as to be 0.01 to 10 units / mL, preferably 0.01 to 5 units / mL.

(3) Phospholipase D
In the present invention, phospholipase D (EC 3.1.4.4) is an enzyme that liberates phosphatidic acid and base by hydrolyzing the ester bond between the phosphatidyl group of glycerophospholipid and the base.

As the phospholipase D used in the present invention, any phospholipase D can be used, for example, derived from plant tissues such as cabbage, carrot, spinach, cottonseed or peanut, derived from animal tissues such as porcine pancreas, or Streptomyces genus And those derived from microorganisms such as Micronospora, Nocardiopsis, Actinomadura or Nocardia.
Examples of the phospholipase D include those prepared by gene recombination technology that incorporates genes such as microorganisms into microorganisms such as Escherichia coli, or phospholipases D prepared from microorganisms whose properties have been improved by gene modification or the like.
In the present invention, phospholipase D derived from Streptomyces microorganisms is preferably used.

The activity value when this phospholipase D is mixed and reacted with the sample and the compound represented by the general formula (I) is preferably in the range of 0.01 to 10 units / mL. A range of 5 units / mL is particularly preferred.
The activity value of phospholipase D contained in the calcium measurement reagent in the sample of the present invention is 0.01 to 10 when the sample and the compound represented by the general formula (I) are mixed and reacted. What is necessary is just to set so that it may become 0.01-5 units / mL so that it may become a unit / mL.
For example, phospholipase D may be contained so as to be 0.01 to 10 units / mL, preferably 0.01 to 5 units / mL.

(4) Substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to calcium to 0.03 to 10 mM (a) Substance that adjusts the apparent Km value to 0.03 to 10 mM The calcium measurement reagent preferably contains a substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to 0.03 to 10 mM.
It is more preferable to include a substance that adjusts the apparent Km value to 0.1 to 3 mM.

In the method for measuring calcium in a sample of the present invention, the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) are mixed and reacted, and the chromogenic group is converted from the compound. In the step of liberating phospholipase C, it is preferable to coexist with a substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to 0.03 to 10 mM.
It is more preferable that a substance that adjusts the apparent Km value to 0.1 to 3 mM coexists.

  A straight line for measuring calcium in a sample by measuring a substance that adjusts the apparent Km value to 0.03 to 10 mM (more preferably 0.1 to 3 mM) in the measurement reagent or coexisting the substance. It is possible to extend the properties to a high concentration range.

Examples of the substance that adjusts the apparent Km value to 0.03 to 10 mM (more preferably 0.1 to 3 mM) include metal ions, chelating agents, amines, basic amino acids, or heterocyclic compounds. Can be mentioned.
That is, the substance that adjusts the apparent Km value to 0.03 to 10 mM (more preferably 0.1 to 3 mM) includes a metal ion, a chelating agent, an amine, a basic amino acid, or a heterocyclic compound. At least one substance selected from the group is contained in the calcium measuring reagent in the sample of the present invention, or is allowed to coexist in the step of releasing the chromophoric group from the substrate in the measuring method of the present invention.
As the substance that adjusts the apparent Km value to 0.03 to 10 mM (more preferably 0.1 to 3 mM), only one type may be used, or a plurality of types may be used simultaneously.
In the present invention, the above metal ions, chelating agents, amines, basic amino acids, or heterocyclic compounds may be used simultaneously.

(B) Metal Ion In the present invention, as a substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to calcium to 0.03 to 10 mM (more preferably 0.1 to 3 mM), a metal ion Can be used.
Examples of the metal ion include cobalt ion, nickel ion, or zinc ion.
Only one type of metal ion may be used, or a plurality of types may be used simultaneously.

In addition, it is preferable that the density | concentration at the time of making this metal ion coexist with the compound represented with a sample, phospholipase C and / or phospholipase D, and said general formula (I) is in the range of 0.005-50 mM. .
For example, the cobalt ion preferably has a range of 0.1 to 50 mM, the nickel ion preferably has a range of 0.05 to 25 mM, and the zinc ion has a range of 0.005 to 2 mM. Preferably it is in the range of 5 mM.

The concentration of the metal ion contained in the calcium measurement reagent in the sample of the present invention is mixed with the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) and reacted. What is necessary is just to set so that the density | concentration in that case may be 0.005-50 mM.
For example, what is necessary is just to contain the said metal ion so that it may become 0.005-50 mM.
As an example, it is preferable to be 0.1 to 50 mM for cobalt ions, 0.05 to 25 mM for nickel ions, and 0 to 0 for zinc ions. It is preferable to be 0.005 to 2.5 mM.

(C) Chelating agent Moreover, in this invention, as a substance which adjusts the apparent Km value with respect to the calcium of said phospholipase C and / or phospholipase D to 0.03-10 mM (preferably 0.1-3 mM), Chelating agents can also be used.

Here, examples of the chelating agent include carboxylic acids such as citric acid, tartaric acid, oxalic acid, malonic acid, fumaric acid, succinic acid, and malic acid, or salts thereof.
Only one type of this chelating agent may be used, or a plurality of types may be used simultaneously.

  In addition, it is preferable that the density | concentration at the time of making this chelating agent coexist with the compound represented with a sample, phospholipase C and / or phospholipase D, and said general formula (I) is in the range of 0.1-200 mM. It is particularly preferable that it is in the range of 5 to 150 mM.

The concentration of the chelating agent contained in the calcium measuring reagent in the sample of the present invention is mixed with the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) and reacted. What is necessary is just to set so that it may become 5 to 150 mM especially so that the density | concentration in that case may be 0.1 to 200 mM.
For example, the chelating agent may be set to be 0.1 to 200 mM, particularly 5 to 150 mM.

(D) Amines, basic amino acids, or heterocyclic compounds In the present invention, the apparent Km value of phospholipase C and / or phospholipase D with respect to calcium is 0.03 to 10 mM (more preferably). Can be used as a substance to be adjusted to 0.1 to 3 mM) amines, basic amino acids, or heterocyclic compounds .

Here, the A Min include, for example, it may be mentioned ammonia, ammonium or derivatives thereof, or salts thereof, and the like. Examples of the ammonium salt include ammonium chloride and ammonium sulfate (ammonium sulfate).
Examples of basic amino acids include arginine, lysine, histidine or a derivative thereof, or a salt thereof.
Moreover, as a heterocyclic compound, imidazole or its derivative (s), or these salts etc. can be mentioned, for example.
In addition, only 1 type may be used for these amines, basic amino acids, or a heterocyclic compound , or multiple types may be used simultaneously.

The concentration when the amines, basic amino acids, or heterocyclic compounds are allowed to coexist with the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) is 1 mM to 2 M. Preferably, it is in the range of 10 mM to 1 M.

In addition, the concentration of the amines, basic amino acids, or heterocyclic compounds contained in the calcium measuring reagent in the sample of the present invention is the sample, phospholipase C and / or phospholipase D, and general formula (1). What is necessary is just to set so that the density | concentration at the time of mixing and making it react with the compound represented may be set to 1 mM-2M, especially 10 mM-1M.
For example, what is necessary is just to set the said amines, basic amino acid, or a heterocyclic compound so that it may become 1 mM-2M, especially 10 mM-1M.

(5) Metal ion The calcium measurement reagent in the sample of the present invention preferably contains a metal ion.
In the method for measuring calcium in a sample of the present invention, the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) are mixed and reacted, and the chromogenic group is converted from the compound. In the step of liberating, it is preferable that a metal ion coexists.

Examples of the metal ions include cobalt ions, nickel ions, and zinc ions.
Only one type of metal ion may be used, or a plurality of types may be used simultaneously.

  By including the metal ion in the measurement reagent or performing the measurement in the coexistence, the linearity of the calcium measurement in the sample can be extended to a high concentration range.

In addition, it is preferable that the density | concentration at the time of making this metal ion coexist with the compound represented with a sample, phospholipase C and / or phospholipase D, and said general formula (I) is in the range of 0.005-50 mM. .
For example, the cobalt ion preferably has a range of 0.1 to 50 mM, the nickel ion preferably has a range of 0.05 to 25 mM, and the zinc ion has a range of 0.005 to 2 mM. Preferably it is in the range of 5 mM.

The concentration of the metal ion contained in the calcium measurement reagent in the sample of the present invention is mixed with the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) and reacted. What is necessary is just to set so that the density | concentration in that case may be 0.005-50 mM.
For example, cobalt ions may be contained in an amount of 0.1 to 50 mM, nickel ions may be contained in an amount of 0.05 to 25 mM, and zinc ions may be contained in an amount of 0 to 50 mM. What is necessary is just to contain so that it may become 0.005-2.5 mM.

(6) Chelating agent The calcium measurement reagent in the sample of the present invention preferably contains a chelating agent.

  In the method for measuring calcium in a sample of the present invention, the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) are mixed and reacted, and the chromogenic group is converted from the compound. In the step of liberating, it is preferable that a chelating agent coexists.

  By including this chelating agent in the measurement reagent or coexisting the measurement, the linearity of the calcium measurement in the sample can be extended to a high concentration range.

  Here, examples of the chelating agent include carboxylic acids such as citric acid, tartaric acid, oxalic acid, malonic acid, fumaric acid, succinic acid, and malic acid, or salts thereof.

  Only one type of this chelating agent may be used, or a plurality of types may be used simultaneously.

  In addition, it is preferable that the density | concentration at the time of making this chelating agent coexist with the compound represented with a sample, phospholipase C and / or phospholipase D, and said general formula (I) is in the range of 0.1-200 mM. It is particularly preferable that it is in the range of 5 to 150 mM.

In addition, the concentration of this chelating agent contained in the calcium measuring reagent in the sample of the present invention is determined by mixing and reacting with the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I). What is necessary is just to set so that it may become 5 to 150 mM especially so that the density | concentration of may be 0.1-200 mM.
For example, the chelating agent may be set to be 0.1 to 200 mM, particularly 5 to 150 mM.

(7) Amines, basic amino acids, or heterocyclic compounds The calcium measurement reagent in the sample of the present invention preferably contains amines, basic amino acids, or heterocyclic compounds .

In the method for measuring calcium in a sample of the present invention, the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) are mixed and reacted, and the chromogenic group is converted from the compound. In the step of liberating, it is preferable that an amine, a basic amino acid, or a heterocyclic compound is allowed to coexist.

By including the amines, basic amino acids, or heterocyclic compounds in the measurement reagent or coexisting the measurement, the linearity of the calcium measurement in the sample can be extended to a high concentration range.

Here, the A Min include, for example, it may be mentioned ammonia, ammonium or derivatives thereof, or salts thereof, and the like. Examples of the ammonium salt include ammonium chloride and ammonium sulfate (ammonium sulfate).
Examples of basic amino acids include arginine, lysine, histidine or a derivative thereof, or a salt thereof.
Moreover, as a heterocyclic compound, imidazole or its derivative (s), or these salts etc. can be mentioned, for example.

In addition, only 1 type may be used for these amines, basic amino acids, or a heterocyclic compound , or multiple types may be used simultaneously.

The concentration when the amines, basic amino acids, or heterocyclic compounds are allowed to coexist with the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) is 1 mM to 2 M. Preferably, it is in the range of 10 mM to 1 M.

The concentration of the amines, basic amino acids, or heterocyclic compounds contained in the calcium measurement reagent in the sample of the present invention is the same as that of the sample, phospholipase C and / or phospholipase D, and general formula (I). What is necessary is just to set so that the density | concentration at the time of mixing and making it react with the compound represented may be set to 1 mM-2M, especially 10 mM-1M.
For example, what is necessary is just to set the said amines, basic amino acid, or a heterocyclic compound so that it may become 1 mM-2M, especially 10 mM-1M.

(8) pH when measuring calcium in the sample
In the method for measuring calcium in a sample of the present invention, the pH at the time of mixing and reacting the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) is pH 5.0 to pH 9. 0.0.

  Further, the pH of the calcium measuring reagent in the sample of the present invention is such that the sample, phospholipase C and / or phospholipase D, and the compound represented by the above general formula (I) are mixed and reacted at pH 5. What is necessary is just to set so that it may become 0-pH9.0.

By the way, when the sample is a body fluid or the like, there is a possibility that phosphodiesterase may be mixed in the sample, and even if calcium is not present in the sample, the phosphodiesterase is represented by the general formula (I). The chromophoric group is liberated from the represented compound, and a signal is generated, giving a positive error to the measured value.
And the optimal pH range of the enzyme activity of this phosphodiesterase is pH 8.0-pH 9.0.

  Therefore, the error due to the phosphodiesterase is set by setting the pH at the time of mixing and reacting to pH 5.5 to pH 8.0, more preferably to pH 5.5 to pH 7.5. Can be suppressed.

In addition, as a buffer solution used so that it may become said pH range, the conventionally well-known buffer solution which has a buffer capacity in the said pH range can be used suitably.
Examples of such buffers include phosphate, tris (hydroxymethyl) aminomethane, imidazole, glycylglycine, MES, Bis-Tris, ADA, ACES, Bis-Tris propane, PIPES, MOPSO, MOPS, BES, Examples of the buffer solutions include HEPES, TES, DIPSO, TAPSO, POPSO, HEPPS, HEPPSO, Tricine, Bicine, and TAPS.

(9) Nonionic surfactant In the method for measuring calcium in the sample of the present invention, in order to stabilize the time course of the reaction, the sample, phospholipase C and / or phospholipase D, and the above general formula (I) When mixing and reacting the compounds represented, it is preferable to coexist with a nonionic surfactant.
For the same reason, it is preferable to include a nonionic surfactant in the calcium measuring reagent in the sample of the present invention.

Examples of such nonionic surfactants include the following.
(A) Polyoxyalkylene such as polyoxyethylene alkyl ether, polyoxypropylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxypropylene alkyl phenyl ether, polyoxyethylene polystyryl phenyl ether, or polyoxyethylene polyoxypropylene glycol Ether compounds.

(B) Polyhydric alcohol partial ester compounds such as glycerin fatty acid partial ester, sorbitan fatty acid partial ester, pentaerythritol fatty acid partial ester, propylene glycol monofatty acid ester, or sucrose fatty acid partial ester.

(C) Polyoxyethylene esterification such as polyoxyethylene sorbitan fatty acid partial ester, polyoxyethylene sorbitol fatty acid partial ester, polyoxyethylene glycerin fatty acid partial ester, polyethylene glycol fatty acid ester, polyglycerin fatty acid partial ester, or polyoxyethylenated castor oil Polyhydric alcohol fatty acid ester.

(D) Amides or amine compounds such as fatty acid diethanolamide, N, N-bis-2-hydroxyalkylamine, polyoxyethylene alkylamine, triethanolamine fatty acid ester, or trialkylamine oxide.

  In addition, as this nonionic surfactant, for example, Triton X-100 (Wako Pure Chemical Industries) or BO-10TX (Nikko Chemicals) is preferable.

  And the density | concentration at the time of making this nonionic surfactant coexist with the compound represented with a sample, phospholipase C and / or phospholipase D, and said general formula (I) is the range of 0.0001 to 1%. It is preferable that it is in the range of 0.001 to 0.5%.

The concentration of the nonionic surfactant contained in the calcium measuring reagent in the sample of the present invention is the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I). What is necessary is just to set so that it may become 0.001-0.5% especially so that the density | concentration at the time of mixing and making it react may be 0.0001-1%.
For example, the above-mentioned nonionic surfactant may be contained in the calcium measuring reagent so as to be 0.0001 to 1%, particularly 0.001 to 0.5%.

(10) Anionic surfactant In the method for measuring calcium in a sample of the present invention, the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) are mixed and reacted. In addition, an anionic surfactant is preferably allowed to coexist.
Further, the calcium measuring reagent in the sample of the present invention preferably contains an anionic surfactant.

Examples of such anionic surfactants include the following.
(A) Aliphatic compound carboxylates such as aliphatic monocarboxylates, N-acyloylsarcosine salts, N-acyloyl-β-alanine salts, or N-acyloyl glutamates; or cyclics such as abietic acid salts Compound carboxylate.

(B) Aliphatic compound sulfonates such as dialkylsulfosuccinate, alkanesulfonate, or hydroxyalkanesulfonate; linear alkylbenzenesulfonate, alkyl (branched) benzenesulfonate, alkylnaphthalenesulfonate , Cyclic sulfonates such as alkylphenoxy polyoxyethylene propyl sulfonate, polyoxyethylene alkyl phenol sulfonate, or naphthalene sulfonate-formaldehyde condensate; or N-methyl-N-oleyl taurine sodium, or N -Nitrogen-containing compound sulfonates such as alkylsulfosuccinic acid monoamide disodium salt.

(C) Sulfated castor oil, sulfated bovine leg oil, fatty acid alkyl ester / sulfate ester salt, alkyl sulfate ester salt, polyoxyethylene alkyl ether sulfate ester, fatty acid monoglyceride sulfate ester, or polyoxyethylene alkyloylamide sulfate An aliphatic compound sulfate such as a polyoxyethylene alkylphenyl ether sulfate, or a cyclic compound sulfate such as a polyoxyethylene styryl phenyl ether sulfate.

(D) Alkyl phosphate ester salts or aliphatic compound phosphate ester salts such as polyoxyethylene alkyl ether phosphate ester salts; or cyclic compound phosphate ester salts such as polyoxyethylene alkylphenyl ether phosphate ester salts.

(E) A saponified polymer compound such as a partially saponified product of styrene-maleic anhydride copolymer or a partially saponified product of olefin-maleic anhydride copolymer.

(F) Polycondensation type polymer compounds such as naphthalene sulfonate-formalin condensate.

(G) Polyoxyethylene alkyl ether acetate, polyoxyethylene alkyl phenyl ether acetate.

  In the method for measuring calcium in a sample of the present invention, an anionic surfactant is coexistent when the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) are mixed and reacted. In addition, by including an anionic surfactant in the calcium measurement reagent of the sample of the present invention, the sensitivity of calcium measurement can be improved, and even a trace amount of calcium in the sample can be accurately measured. You will be able to make measurements.

Furthermore, in the method for measuring calcium in a sample of the present invention, an anionic surfactant is used when the sample, phospholipase C and / or phospholipase D, and the compound represented by the above general formula (I) are mixed and reacted. In addition, the enzyme activity of phosphodiesterase can be suppressed by including an anionic surfactant in the calcium measuring reagent in the sample of the present invention. Even if phosphodiesterase is mixed, errors in measured values derived from this phosphodiesterase can be prevented.
That is, by making this anionic surfactant coexist or containing, even if the pH is not set to pH 5.5 to pH 8.0 (more preferably pH 5.5 to pH 7.5) as described above, An error caused by phosphodiesterase can be prevented.

In order to improve the sensitivity of calcium measurement by coexisting or including this anionic surfactant, examples of the anionic surfactant include sulfonate compounds, polyoxyethylene alkyl ether sulfates. A salt, an N-acyl taurine salt or the like is preferable.
More specifically, for example, dioctyl sodium sulfosuccinate (OTP-100) [Nikko Chemicals Co., Ltd.] is used as the sulfonate compound, and SBL-2N-27 [Nikko Chemicals is used as the polyoxyethylene alkyl ether sulfate ester salt. And N-acyl taurine salts include CMT-30 or LMT [Nikko Chemicals Co., Ltd.].

In addition, by coexisting or including this anionic surfactant, to suppress the enzyme activity of phosphodiesterase, to prevent errors in measured values derived from phosphodiesterase mixed in the sample, Examples of the anionic surfactant include N-acyloyl sarcosine salt, polyoxyethylene alkylphenyl ether sulfate ester salt, polyoxyethylene alkyl ether acetate salt, cholic acid or its salt, or deoxycholic acid or its salt, etc. Is preferred.
More specifically, for example, as this N-acylyl sarcosine salt, cocoyl sarcosine sodium (sarcosinate CN-30) [Nikko Chemicals Co., Ltd.] or lauroyl sarcosine sodium (sarcosinate LN) [Nikko Chemicals Co., Ltd.] or the like is used. Examples of the alkyl phenyl ether sulfate ester salt include polyoxyethylene nonyl phenyl ether sulfate triethanolamine (SNP-4T) [Nikko Chemicals Co., Ltd.] and the like, and examples of the polyoxyethylene alkyl ether acetate salt include sodium polyoxyethylene alkyl ether acetate (ECTD- 6NEX) [Nikko Chemicals Co., Ltd.], sodium cholate as the cholate, and sodium deoxycholate as the deoxycholate.

  The concentration when the anionic surfactant coexists with the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) is in the range of 0.0001 to 1%. It is preferable that it is in the range of 0.001 to 0.5%.

The concentration of the anionic surfactant to be contained in the calcium measuring reagent in the sample of the present invention is the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I). What is necessary is just to set so that it may become 0.001-0.5% especially so that the density | concentration at the time of mixing and making it react may be 0.0001-1%.
For example, the anionic surfactant may be set to 0.0001 to 1%, particularly 0.001 to 0.5%.

(11) Addition of calcium In the calcium measurement reagent or the calcium measurement method in the sample of the present invention, calcium ions or a salt thereof may be added to the measurement reagent or the measurement reaction solution and coexist if necessary. Good.
By adding and coexisting calcium ions or salts thereof, it is possible to improve the phenomenon of calibration curve penetration in the measurement of calcium in a sample.

(12) Constituent components such as measuring reagent In the calcium measuring reagent or calcium measuring method in the sample of the present invention, in addition to the above components, known antiseptics such as antibacterial agents such as caisson CG or antibiotic agents such as gentamicin An agent or the like can be appropriately included or allowed to coexist as necessary.

(13) Configuration of Reagents The calcium measurement method and the calcium measurement reagent in the sample of the present invention can be implemented and configured by a one-step method (one reagent system). It is more preferable from the viewpoint of the stability of the measurement reagent to implement and configure by a multi-step method (multi-reagent system) such as a 2-step method (2-reagent system).

The phospholipase C and / or phospholipase D and the compound represented by the general formula (I) are preferably contained in separate measuring reagents.
This is because the compound represented by the above general formula (I) is decomposed by phospholipase C and / or phospholipase D while it is stored for a long time when calcium is mixed even in a trace amount. is there.

(14) Enzyme acting on phosphate monoester compound In the method for measuring calcium in the sample of the present invention, when X2 of the compound represented by the general formula (I) is a chromophoric group having a nitrophenyl group , When the sample, phospholipase C and / or phospholipase D, and the compound represented by the general formula (I) are mixed and reacted, the ester bond of the phosphate monoester compound does not act on the phosphate diester compound. It is preferable to coexist an enzyme that hydrolyzes.

When calcium is present in the sample, phospholipase C and / or phospholipase D hydrolyzes and releases X 2 together with X 1 of the compound represented by the general formula (I). Depending on the conditions, it may take time from hydrolysis of the chromophoric group having the first nitrophenyl group to hydrolysis of the chromophoric group having the second nitrophenyl group .

At this time, if there is an enzyme that hydrolyzes the ester bond of the phosphate monoester compound without acting on the phosphate diester compound, it is generated by hydrolysis of the chromophoric group having the first nitrophenyl group. An enzyme that hydrolyzes the ester bond of the phosphoric acid monoester compound without acting on the phosphoric acid diester compound acts on the phosphoric acid monoester compound, and a chromophoric group having a second nitrophenyl group This is because can be hydrolyzed in a short time.

Since the enzyme does not act on the phosphodiester compound, calcium does not exist in the sample, and phospholipase C and / or phospholipase D does not act on the compound represented by the general formula (I) (hydrolysis). If no degradation), this enzyme also does not hydrolyze a chromogenic group that having a nitrophenyl group and does not cause a positive error in the measurement value.

For the same reason, in the calcium measuring reagent in the sample of the present invention, when X2 of the compound represented by the general formula (I) is a chromophoric group having a nitrophenyl group, it acts on a phosphodiester compound. It is preferable to include an enzyme that hydrolyzes the ester bond of the phosphate monoester compound.

Examples of the enzyme that hydrolyzes the ester bond of the phosphate monoester compound without acting on the phosphate diester compound include alkaline phosphatase (ALP) and acid phosphatase (ACP).

An enzyme that hydrolyzes the ester bond of the phosphate monoester compound without acting on the phosphate diester compound is a sample, phospholipase C and / or phospholipase D, and a compound represented by the above general formula (I) It is preferable that the activity value at the time of mixing and making it react with is in the range of 0.01 to 10 units / mL, particularly preferably in the range of 0.1 to 5 units / mL.

Also, to be contained in the calcium assay reagent in a sample of the present invention, activity of enzyme which hydrolyzes an ester bond of phosphoric acid monoester compound does not act on the phosphoric acid diester compound, sample, phospholipase C and / Alternatively, the activity value when mixed and reacted with the phospholipase D and the compound represented by the general formula (1) is preferably 0.01 to 5 units / mL. Should be set to be.
For example, the enzyme that hydrolyzes the ester bond of the phosphoric acid monoester compound without acting on this phosphoric acid diester compound is preferably 0.01 to 5 units / mL so as to be 0.01 to 10 units / mL. What is necessary is just to contain so that it may become mL.

(15) An example of measuring calcium in a sample The method for measuring calcium in a sample of the present invention is as follows.
a) A sample, phospholipase C and / or phospholipase D, and a compound represented by the above general formula (I) as a substrate of phospholipase C and / or phospholipase D are mixed and reacted, and a nitrophenyl group is reacted from the compound. And b) a step of colorimetrically measuring the chromophoric group having the liberated nitrophenyl group .

  The method for measuring calcium in a sample of the present invention is characteristic in that the measurement can be performed without intervening a conjugate enzyme such as choline oxidase, alkaline phosphatase, or acid phosphatase.

  An example of the case of measuring calcium in a sample using the method and reagent for measuring calcium in a sample of the present invention will be described more specifically. For example, calcium contained in a sample and phospholipase C and / or phospholipase D Phospholipase C and / or phospholipase D is activated by this calcium.

When this phospholipase C and / or phospholipase D activated by calcium is brought into contact with the compound represented by the general formula (I), the phospholipase C and / or phospholipase D is represented by the general formula (I). The chromophoric group (X1, optionally further X2) having a nitrophenyl group of the resulting compound is hydrolyzed and released.

That is, a chromogenic group having a nitrophenyl group such as a 4-nitrophenyl group, a 2-chloro-4-nitrophenyl group, or a 2,6-dichloro-4-nitrophenyl group is released.

Here liberated 4-nitrophenol, 2-chloro-4-nitrophenol, or 2,6-dichloro-4-nitrophenol or the like in any wavelength showing the absorption, and the absorbance using a spectrophotometer The amount of the chromophoric group having a liberated nitrophenyl group is determined by measurement, and the activity value of phospholipase C and / or phospholipase D is determined therefrom. Further, the calcium concentration in the sample can be calculated by proportional calculation with the absorbance when a standard solution with a known calcium concentration is measured.

  The measurement principle of the present invention is as follows when bis (4-nitrophenyl) phosphoric acid is used as an example of the compound represented by the general formula (I).

  In the present invention, when bis (4-nitrophenyl) phosphate is used as a substrate as the compound represented by the general formula (I), phospholipase C and / or phospholipase D activated by calcium ions in the sample. Each hydrolyzes the ester bond between the phosphate group of bis (4-nitrophenyl) phosphate and two 4-nitrophenyl groups to liberate two molecules of 4-nitrophenol.

  By measuring the absorbance of the released 4-nitrophenol using a spectrophotometer or the like at a measurement wavelength of 405 nm or the like, the calcium concentration in the sample can be measured.

(16) Sample In the present invention, a sample may contain calcium, calcium ions or a salt thereof, and is intended to measure the calcium concentration in the sample. If it is, it will not specifically limit.
Examples of such a sample include human or animal blood, serum, plasma, urine, spinal fluid, saliva, sweat, and other body fluids, human or animal kidneys, heart, lung, brain and other organ extracts. Extracts such as skeletal muscle, bone marrow, skin, or nerve tissue; extracts such as hair; human or animal fecal extracts or suspensions; cell extracts; plant extracts; foods or extracts thereof Agricultural, forestry and fishery products or extracts thereof; drinking water; beverages; environmental samples (soil, seawater, river water, lake water, groundwater, etc.); or drugs.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by this Example.

(Measurement of calcium in sample)
The calcium concentration in the sample was measured by the calcium measuring reagent and the calcium measuring method in the sample of the present invention.

1. Preparation of Reagent (1) Preparation of First Reagent The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to 6.5 (20 ° C.) to prepare a first reagent.
PIPES (Oriental Yeast Co., Ltd.) 80 mM
BO-10TX (Nikko Chemicals) 0.1%
Caisson CG (Rohm and Haas) 0.05%
Nickel acetate 5 mM
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei) 2 units / mL

(2) Preparation of Second Reagent A The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 6.5 (20 ° C.) to prepare the second reagent A.
PIPES (Oriental Yeast Co., Ltd.) 5mM
Bis (4-nitrophenyl) phosphate [substrate] (Wako Pure Chemical Industries, Ltd.) 24 mM

(3) Preparation of second reagent B The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 6.5 (20 ° C.) to prepare second reagent B.
PIPES (Oriental Yeast Co., Ltd.) 5mM
4-Nitrophenylphenyl phosphate [substrate] (Sigma) 10 mM

(4) Preparation of Second Reagent C The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 6.5 (20 ° C.) to prepare the second reagent C.
PIPES (Oriental Yeast Co., Ltd.) 5mM
4-Nitrophenyl phosphate [substrate] (Daiichi Chemical Co., Ltd.) 96 mM

2. Preparation of Sample Sample containing calcium of the following concentration by diluting a calcium standard solution [0.1M nitric acid aqueous solution containing 1,000 mg / mL calcium carbonate; for atomic absorption] (Wako Pure Chemical Industries, Ltd.) with pure water. Were prepared respectively.

・ 20.0mg / dL
・ 17.5mg / dL
・ 15.0mg / dL
・ 12.5mg / dL
・ 10.0mg / dL
・ 7.5mg / dL
・ 5.0mg / dL
・ 2.5mg / dL

The pure water was a sample not containing calcium, that is, a “sample with a calcium concentration of 0 mg / dL”.
Nine types of samples were prepared as described above.

3. Measurement of Calcium Concentration in Sample The calcium concentration in the sample was measured with a 7170S type automatic analyzer manufactured by Hitachi, Ltd.

(1) Nine types of samples prepared in 2 above were used as samples, and 180 μL of the first reagent A prepared in 1 (1) above was added to 6 μL of the sample, and the mixture was reacted at 37 ° C. for 5 minutes.

(2) Thereafter, 60 μL of the second reagent A prepared in (1) above was added thereto and reacted at 37 ° C.

(3) Then, the absorbance at the main wavelength of 405 nm and the sub wavelength of 660 nm was measured from 1 minute 55 seconds (23 point) to 5 minutes 8 seconds (34 point) after the addition of the second reagent A.

(4) The absorbance change per minute (ΔAbs. / Min) was calculated from the absorbance measured in (3) above.

(5) In addition, except that the second reagent B prepared in (1) above is used instead of the second reagent A, the operation is performed as in the above (1) to (4), and the operation is performed per minute. The amount of change in absorbance (ΔAbs. / Min) was determined.

(6) Further, except that the second reagent C prepared in the above (4) is used instead of the second reagent A, the operation is performed as in the above (1) to (4), and the operation is performed per minute. The amount of change in absorbance (ΔAbs. / Min) was determined.

4). Measurement Results The above measurement results are shown in FIG.
In this figure, the horizontal axis represents the calcium concentration (mg / dL) in the sample, and the vertical axis represents the change in absorbance per minute (ΔAbs. / Min) obtained by measurement.

As is apparent from this figure, it can be seen that each absorbance change amount (ΔAbs. / Min) increases almost linearly as the calcium concentration in the sample increases.
From this, it was confirmed that calcium in the sample can be quantitatively measured.

  In addition, when bis (4-nitrophenyl) phosphoric acid is used as a substrate, the signal (absorbance change) obtained compared to when 4-nitrophenylphenylphosphoric acid or 4-nitrophenylphosphoric acid is used. Is very high.

  Therefore, when bis (4-nitrophenyl) phosphoric acid is used as a substrate, that is, when both X1 and X2 are chromogenic groups in the compound of the general formula (I), measurement is highly sensitive. We were able to confirm that

(Confirmation of linearity improvement effect by metal ions)
In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, the linearity improving effect was confirmed when metal ions were contained in the measuring reagent.

1. Preparation of Reagent (1) Preparation of Cobalt Ion-Containing First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, and the pH is adjusted to 6.5 (20 ° C.). One reagent was prepared.
PIPES (Oriental Yeast Co., Ltd.) 80 mM
BO-10TX (Nikko Chemicals) 0.1%
Caisson CG (Rohm and Haas) 0.05%
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei) 2 units / mL
Cobalt chloride 16 mM, 8 mM, 4 mM, 2 mM, 1 mM, or not contained (0 mM)

(2) Preparation of nickel ion-containing first reagent Six types having the same components and concentrations as the cobalt ion-containing first reagent of (1), except that each contains (or does not contain) nickel acetate at the following concentrations. The first reagent was prepared.

・ 8 mM
・ 4 mM
・ 2 mM
・ 1 mM
・ 0.5 mM
・ 0 mM

(3) Preparation of zinc ion-containing first reagent Six components having the same components and concentrations as the cobalt ion-containing first reagent of (1), except that each contains (or does not contain) the following concentrations of zinc acetate: The first reagent was prepared.

・ 0.8 mM
・ 0.4 mM
・ 0.2 mM
・ 0.1 mM
・ 0.05 mM
・ 0 mM

(4) Second reagent The second reagent A prepared in (1) of Example 1 was used as the second reagent.

2. Sample Preparation Nine types of samples prepared in 2 of Example 1 were used as samples.

3. Measurement of calcium concentration in sample 3 of Example 1 except that the first reagent prepared in (1), (2) and (3) above was used instead of the first reagent. (1) to (4) were performed, and the change in absorbance per minute (ΔAbs. / Min) was determined.

(4) Measurement results Fig. 2 shows the measurement results when cobalt ions are contained in the measurement reagent, Fig. 3 shows the measurement results when nickel ions are contained, and the measurement results when zinc ions are contained. Is shown in FIG.

  In this figure, the horizontal axis represents the calcium concentration (mg / dL) in the sample, and the vertical axis represents the change in absorbance per minute (ΔAbs. / Min) obtained by measurement.

The apparent Km value in each case is shown in Table 1.
The apparent Km value was calculated from the slope with Vmax fixed in the Lineweaver-Burk plot.

From the above measurement results, the following can be understood.
When cobalt ions are contained in the measurement reagent, the relationship [calibration curve] between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) becomes more linear than when no reagent is contained. It was confirmed that the linearity was improved. And it turns out that the quantitative property of a measurement is improving by this.

  In addition, when nickel ions are contained in the measurement reagent, the relationship [calibration curve] between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) becomes more linear than when no nickel ions are contained. It was confirmed that the linearity of the line was improved. And it turns out that the quantitative property of a measurement is improving by this.

  Furthermore, when zinc ions are contained in the measurement reagent, the relationship [calibration curve] between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) becomes more linear than when no zinc ions are contained. It was confirmed that the linearity of the line was improved. And it turns out that the quantitative property of a measurement is improving by this.

  In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, the linearity improvement effect when citric acid as a chelating agent was contained in the measuring reagent was confirmed.

1. Preparation of Reagent (1) Preparation of First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, and the pH is adjusted to 7.0 (20 ° C.). Prepared.
PIPES (Oriental Yeast Co., Ltd.) 80 mM
BO-10TX (Nikko Chemicals) 0.1%
Caisson CG (Rohm and Haas) 0.05%
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei Corporation) 1 unit / mL
Citric acid 4 mM, 2 mM, 1 mM, 0.5 mM, or not contained (0 mM)

(2) Second reagent The second reagent A prepared in (1) of Example 1 was used as the second reagent.

2. Preparation of Sample Sample containing calcium of the following concentration by diluting a calcium standard solution [0.1M nitric acid aqueous solution containing 1,000 mg / mL calcium carbonate; for atomic absorption] (Wako Pure Chemical Industries, Ltd.) with pure water. Were prepared respectively.

・ 20.0mg / dL
・ 15.0mg / dL
・ 10.0mg / dL
・ 5.0mg / dL
・ 2.5mg / dL

The pure water was a sample not containing calcium, that is, a “sample with a calcium concentration of 0 mg / dL”.
The above six types of samples were prepared.

3. Measurement of the calcium concentration in the sample Using the first reagent prepared in (1) above in place of the first reagent, respectively, and using the six types of samples prepared in 2 above as samples. Except for the above, operations were performed as in (1) to (4) of 3 of Example 1, and the amount of change in absorbance per minute (ΔAbs. / Min) was determined.

4). Measurement Results The measurement results are shown in FIG.

  In this figure, the horizontal axis represents the calcium concentration (mg / dL) in the sample, and the vertical axis represents the change in absorbance per minute (ΔAbs. / Min) obtained by measurement.

This figure shows the following.
When citric acid, which is a chelating agent, is included in the measurement reagent, the relationship [calibration curve] between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) becomes more linear than when it is not included. It was confirmed that the linearity of the calibration curve was improved. And it turns out that the quantitative property of a measurement is improving by this.

(Confirmation of linearity improvement effect by chelating agent-2)
In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, the linearity improving effect when oxalic acid, tartaric acid or citric acid as a chelating agent was contained in the measuring reagent was confirmed.

1. Preparation of Reagent (1) Preparation of Oxalic Acid-Containing First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, pH is adjusted to 6.5 (20 ° C), and oxalic acid is contained. A first reagent was prepared.
PIPES (Oriental Yeast Co., Ltd.) 80 mM
BO-10TX (Nikko Chemicals) 0.1%
Caisson CG (Rohm and Haas) 0.05%
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei Corporation) 0.66 unit / mL
Oxalic acid 5 mM

(2) Preparation of Tartaric Acid-Containing First Reagent A tartaric acid-containing first reagent having the same components and concentration as the oxalic acid-containing first reagent of (1) except that 20 mM tartaric acid is contained instead of 5 mM oxalic acid. One reagent was prepared.

(3) Preparation of citric acid-containing first reagent Citric acid having the same components and concentration as the oxalic acid-containing first reagent of (1) except that 1 mM citric acid is contained instead of 5 mM oxalic acid. A first reagent containing was prepared.

(4) Second reagent The second reagent A prepared in (1) of Example 1 was used as the second reagent.

2. Preparation of Sample Sample containing calcium of the following concentration by diluting a calcium standard solution [0.1M nitric acid aqueous solution containing 1,000 mg / mL calcium carbonate; for atomic absorption] (Wako Pure Chemical Industries, Ltd.) with pure water. Were prepared respectively.

・ 20.0mg / dL
・ 10.0mg / dL
・ 5.0mg / dL
・ 2.5mg / dL

The pure water was a sample not containing calcium, that is, a “sample with a calcium concentration of 0 mg / dL”.
The above five types of samples were prepared.

3. Measurement of calcium concentration in sample Using each of the three types of first reagents prepared in (1), (2) and (3) above instead of the first reagent, and preparing in 2 above as samples Except for using a total of 5 types of samples, the operation was performed as in 3 (1) to (4) of Example 1 to determine the amount of change in absorbance per minute (ΔAbs. / Min).

4). Measurement Results The measurement results are shown in FIG.

  In this figure, the horizontal axis represents the calcium concentration (mg / dL) in the sample, and the vertical axis represents the change in absorbance per minute (ΔAbs. / Min) obtained by measurement.

This figure shows the following.
When the chelating agent oxalic acid, tartaric acid, or citric acid is contained in the measurement reagent, the relationship [calibration curve] between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) becomes almost linear, It was confirmed that the linearity of the calibration curve was improved. And it turns out that the quantitative property of a measurement is improving by this.

(Confirmation of time course stabilization effect of reaction with nonionic surfactant)
In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, the time course stabilizing effect of the reaction when BO-10TX, which is a nonionic surfactant, was contained in the measuring reagent was confirmed.

1. Preparation of Reagent (1) Preparation of First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, and the pH is adjusted to 6.5 (20 ° C.). Prepared.
PIPES (Oriental Yeast Co., Ltd.) 80 mM
Caisson CG (Rohm and Haas) 0.05%
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei) 2 units / mL
BO-10TX (Nikko Chemicals) 0.10%, 0.01%, 0.001%, 0.0001%, or not included (0%)

(2) Second reagent The second reagent A prepared in (1) of Example 1 was used as the second reagent.

2. Preparation of sample Calcium standard solution [0.1 M nitric acid aqueous solution containing 1,000 mg / mL calcium carbonate; for atomic absorption] (Wako Pure Chemical Industries, Ltd.) was diluted with pure water to obtain a calcium concentration of 10.0 mg / dL. Samples were prepared.

3. Confirmation of reaction time course The time course (progress) of the calcium concentration measurement reaction in the sample was confirmed with a Hitachi 7170S automatic analyzer.

(1) To 6 μL of the sample prepared in 2 above, 180 μL of each of the 5 types of first reagents prepared in 1 (1) above was added and reacted at 37 ° C. for 5 minutes after mixing.

(2) Thereafter, 60 μL of the second reagent A prepared in (1) above was added thereto and reacted at 37 ° C.

(3) Then, the absorbance at the main wavelength of 405 nm and the sub wavelength of 660 nm was measured from the time of mixing the sample and the first reagent, and the reaction time course was recorded.

4). Measurement Results The measurement results are shown in FIG.

  In this figure, the horizontal axis represents the elapsed time after mixing the sample and the first reagent, and the vertical axis represents the absorbance (Abs.) Obtained by the measurement.

This figure shows the following.
When BO-10TX, which is a nonionic surfactant, is included in the measurement reagent, the progress (time course) of the reaction is uniform and linear compared to when it is not included, and the quantitativeness of the measurement is improved. It was confirmed that he was doing.

(Confirmation of sensitivity improvement effect by anionic surfactant)
In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, the effect of improving the measurement sensitivity when LMT, which is an anionic surfactant, was contained in the measuring reagent was confirmed.

1. Preparation of Reagent (1) Preparation of First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, and the pH is adjusted to 7.25 (20 ° C.). Prepared.
PIPES (Oriental Yeast Co., Ltd.) 80 mM
BO-10TX (Nikko Chemicals) 0.1%
Caisson CG (Rohm and Haas) 0.05%
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei Corporation) 1 unit / mL
LMT (Nikko Chemicals) 0.1%, 0.05%, 0.025%, or not included (0%)

(2) Preparation of Second Reagent The following reagent components were dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 6.5 (20 ° C.) to prepare a second reagent.
PIPES (Oriental Yeast Co., Ltd.) 5mM
Bis (4-nitrophenyl) phosphate [substrate] (Wako Pure Chemical Industries, Ltd.) 1 mM
Citric acid 12 mM

2. Preparation of Sample Sample containing calcium of the following concentration by diluting a calcium standard solution [0.1M nitric acid aqueous solution containing 1,000 mg / mL calcium carbonate; for atomic absorption] (Wako Pure Chemical Industries, Ltd.) with pure water. Were prepared respectively.

・ 20.0mg / dL
・ 15.0mg / dL
・ 10.0mg / dL
・ 5.0mg / dL
・ 2.5mg / dL

The pure water was a sample not containing calcium, that is, a “sample with a calcium concentration of 0 mg / dL”.
The above six types of samples were prepared.

(3) Measurement of calcium concentration in sample Using each of the four types of first reagents prepared in (1) above instead of the first reagent, and using (1) (2) above instead of the second reagent A Except for using the second reagent prepared in (2) and using a total of six types of samples prepared in (2) above as the samples, the operation was performed as in (1) to (4) of 3 in Example 1, The amount of change in absorbance per minute (ΔAbs. / Min) was determined.

4). Measurement Results The measurement results are shown in FIG.

  In this figure, the horizontal axis represents the calcium concentration (mg / dL) in the sample, and the vertical axis represents the change in absorbance per minute (ΔAbs. / Min) obtained by measurement.

This figure shows the following.
When LMT, which is an anionic surfactant, is contained in the measurement reagent, the amount of change in absorbance (signal) per minute obtained is significantly higher than when LMT is not contained, and the sensitivity of measurement is increased. It was confirmed to rise. From this, it was confirmed that even a trace amount of calcium could be measured.

(Confirmation of phosphodiesterase activity inhibitory effect by anionic surfactant)
In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, the effect of suppressing the phosphodiesterase activity when an anionic surfactant was allowed to coexist during the measurement reaction was confirmed.

1. Preparation of Reagent (1) Preparation of First Reagent The following reagent components were dissolved in pure water so as to have the stated concentrations, respectively, and the pH was adjusted to 7.75 (20 ° C.) to prepare a first reagent.
HEPES (Oriental Yeast Co., Ltd.) 60 mM
Triton X-100 (Nikko Chemicals) 0.1%
Caisson CG (Rohm and Haas) 0.05%
Bis (4-nitrophenyl) phosphate [substrate] (Wako Pure Chemical Industries, Ltd.) 4 mM

(2) Preparation of Second Reagent Each of the 31 types of surfactants shown in FIG. 9 was dissolved in pure water to a concentration of 5% to prepare 31 types of second reagents.

(3) Preparation of control / second reagent Pure water was used as the control / second reagent.

2. Sample preparation Human serum and physiological saline (0.9% sodium chloride aqueous solution) were used as samples.

3. Measurement of sample blank value (serum blank value) The sample blank value (serum blank value) was measured using a Hitachi 7150 type automatic analyzer.

(1) 180 μL of the first reagent prepared in (1) above was added to 10 μL of the above 2 human serum sample, and after mixing, the mixture was reacted at 37 ° C. for 5 minutes.

(2) Then, 20 μL of the second reagent prepared in (1) above was added to this and reacted at 37 ° C.

(3) Then, the absorbance at the main wavelength of 405 nm and the sub wavelength of 660 nm was measured from 2 minutes 58 seconds (40 points) to 4 minutes 57 seconds (50 points) after the addition of the second reagent.

(4) The absorbance change per minute (ΔAbs. / Min) was calculated from the absorbance measured in (3) above.

(5) The change in absorbance per minute (ΔAbs. /) Is performed by performing the operations as described in (1) to (4) except that the physiological saline of 2 is used as a sample instead of human serum. Min).

(6) The sample blank is obtained by subtracting the absorbance change per minute (ΔAbs. / Min) determined in (5) from the absorbance change per minute (ΔAbs. / Min) determined in (4) above. The value (serum blank value) was determined.

(7) In all 31 types of second reagents prepared in (1) above, the operations of (1) to (6) were performed, and each sample blank value (serum blank value) was determined. Further, the control sample blank value (serum blank value) was determined in the same manner by replacing the second reagent with the control / second reagent of the above (3).

4). Measurement Results The above measurement results are shown in FIG.
In this figure, the horizontal axis represents the amount of change in absorbance (ΔAbs. / Min) when the measurement reagent does not contain phospholipase C and / or phospholipase D and does not cause the calcium measurement reaction of the present invention, that is, the sample blank value. (Serum blank value) is represented, and the vertical axis represents the surfactant used in the second reagent.

From this figure, in the nonionic surfactants from BD-6SY to BPS-30, the sample blank value (serum blank value) exceeds 10 × 10 ⁴ (ΔAbs. / Min), and the reaction solution contains It can be seen that despite the absence of phospholipase C and / or phospholipase D, a reaction in which 4-nitrophenol is liberated from bis (4-nitrophenyl) phosphate as a substrate occurs. That is, it can be seen that 4-nitrophenol is liberated from the substrate by phosphodiesterase mixed in the serum sample, and a positive error occurs in the measured value.

On the other hand, in the case of cholic acid, ECTD-6NEX, sodium deoxycholate, SNP-4N, sarcosinate LN, and sarcosinate CN-30, which are anionic surfactants, the sample blank value (serum blank value) Is 10 × 10 ⁴ (ΔAbs. / Min) or less, and the reaction of liberating 4-nitrophenol from bis (4-nitrophenyl) phosphate, which is a substrate, is suppressed. It can be seen that the activity of diesterase is suppressed.
From the above, by containing or coexisting an anionic surfactant in the measurement reagent or measurement reaction, the activity of phosphodiesterase mixed in the serum sample is suppressed, resulting in a positive error in the measured value. It was confirmed that it can be prevented.

(Confirmation of linearity improvement effect by nitrogen-containing compound-1)
In the calcium measuring reagent and calcium measuring method in the sample of the present invention, the linearity improving effect was confirmed when ammonium sulfate (ammonium sulfate), which is a nitrogen-containing compound, was contained in the measuring reagent.

1. Preparation of Reagent (1) Preparation of First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, pH is adjusted to 7.75 (20 ° C.), and six types of first reagent are added. Prepared.
HEPES (Oriental Yeast Co., Ltd.) 60 mM
Triton X-100 (Nikko Chemicals) 0.1%
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei Corporation) 0.5 units / mL
Sarcosinate LN (Nikko Chemicals) 0.5%
Ammonium sulfate (ammonium sulfate) 1M, 0.8M, 0.6M, 0.4M, 0.2M, or not contained (0M)

(2) Second Reagent The following reagent components were dissolved in pure water so as to have the respective concentrations described above, pH was adjusted to 7.75 (20 ° C.), and six types of second reagents were prepared.
HEPES (Oriental Yeast Co., Ltd.) 60 mM
Triton X-100 (Nikko Chemicals) 0.1%
Bis (4-nitrophenyl) phosphate [substrate] (Wako Pure Chemical Industries, Ltd.) 4 mM
Sarcosinate LN (Nikko Chemicals) 0.5%
Ammonium sulfate (ammonium sulfate) 1M, 0.8M, 0.6M, 0.4M, 0.2M, or not contained (0M)

2. Preparation of Sample Sample containing calcium of the following concentration by diluting a calcium standard solution [0.1M nitric acid aqueous solution containing 1,000 mg / mL calcium carbonate; for atomic absorption] (Wako Pure Chemical Industries, Ltd.) with pure water. Were prepared respectively.

・ 40.0mg / dL
・ 35.0mg / dL
・ 30.0mg / dL
・ 25.0mg / dL
・ 20.0mg / dL
・ 15.0mg / dL
・ 10.0mg / dL
・ 5.0mg / dL
・ 2.5mg / dL

The pure water was a sample not containing calcium, that is, a “sample with a calcium concentration of 0 mg / dL”.
The above 10 types of samples were prepared.

3. Measurement of Calcium Concentration in Sample The sample was measured with a 7150 type automatic analyzer manufactured by Hitachi, Ltd.

(1) To 9 μL of the sample prepared in 2 above, 180 μL of the first reagent prepared in 1 (1) above was added, and after mixing, reacted at 37 ° C. for 5 minutes.

(2) Thereafter, 180 μL of the second reagent prepared in (1) above was added thereto and reacted at 37 ° C. (In addition, the 1st reagent and the 2nd reagent each used the thing of the same ammonium sulfate density | concentration.)

(3) Then, the absorbance at the main wavelength of 405 nm and the sub wavelength of 660 nm was measured from 2 minutes 58 seconds (40 points) to 4 minutes 57 seconds (50 points) after the addition of the second reagent.

(4) The absorbance change per minute (ΔAbs. / Min) was calculated from the absorbance measured in (3) above.

4). Measurement Results The above measurement results are shown in FIG.

  In this figure, the horizontal axis represents the calcium concentration (mg / dL) in the sample, and the vertical axis represents the change in absorbance per minute (ΔAbs. / Min) obtained by measurement.

This figure shows the following.
When the measurement reagent contains ammonium sulfate (ammonium sulfate), which is a nitrogen-containing compound, the relationship [calibration curve] between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) is greater than when it is not contained. It became linear, and it was confirmed that the linearity of the calibration curve was improved. And it turns out that the quantitative property of a measurement is improving by this.

(Confirmation of linearity improvement effect by nitrogen-containing compound -2)
In the calcium measuring reagent and the calcium measuring method in the sample of the present invention, the linearity improving effect was confirmed when arginine (basic amino acid) or imidazole (heterocyclic compound), which is a nitrogen-containing compound, was contained in the measuring reagent. .

1. Preparation of Reagent (1) Preparation of Arginine-Containing First Reagent The following reagent components are dissolved in pure water so that each concentration is as described, and the pH is adjusted to 7.0 (20 ° C.). Reagents were prepared.
HEPES (Oriental Yeast Co., Ltd.) 60 mM
Triton X-100 (Nikko Chemicals) 0.1%
LMT (Nikko Chemicals) 0.5%
Calcium acetate 0.15 mM
Phospholipase D [derived from Streptomyces chromofuscus] (Asahi Kasei Corporation) 0.75 unit / mL
Arginine (Wako Pure Chemical Industries, Ltd.) 1M, 0.75M, 0.5M, 0.25M, or not contained (0M)

(2) Preparation of imidazole-containing first reagent Arginine-containing first reagent of the above (1), components and concentrations, except that each arginine contains (or does not contain) imidazole (Hanai Chemical Co.) at the following concentrations: Five first reagents having the same value were prepared.

・ 1M
・ 0.75M
・ 0.5M
・ 0.25M
・ 0M

(3) Second Reagent The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a second reagent.
HEPES (Oriental Yeast Co., Ltd.) 60 mM
Triton X-100 (Nikko Chemicals) 0.1%
Bis (4-nitrophenyl) phosphate [substrate] (Wako Pure Chemical Industries, Ltd.) 6 mM

2. Preparation of Sample Sample containing calcium of the following concentration by diluting a calcium standard solution [0.1M nitric acid aqueous solution containing 1,000 mg / mL calcium carbonate; for atomic absorption] (Wako Pure Chemical Industries, Ltd.) with pure water. Were prepared respectively.

・ 20.0mg / dL
・ 17.5mg / dL
・ 15.0mg / dL
・ 12.5mg / dL
・ 10.0mg / dL
・ 7.5mg / dL
・ 2.5mg / dL

The pure water was a sample not containing calcium, that is, a “sample with a calcium concentration of 0 mg / dL”.
The above 8 types of samples were prepared.

3. Measurement of Calcium Concentration in Sample The sample was measured with a 7150 type automatic analyzer manufactured by Hitachi, Ltd.

(1) 240 μL of the arginine-containing first reagent prepared in (1) above was added to 9 μL of the sample prepared in 2 above, and the mixture was reacted at 37 ° C. for 5 minutes after mixing.

(2) Thereafter, 120 μL of the second reagent prepared in the above (3) was added thereto and reacted at 37 ° C.

(3) Then, the absorbance at the main wavelength of 405 nm and the sub wavelength of 660 nm was measured from 1 minute 47 seconds (34 points) to 4 minutes 57 seconds (50 points) after the addition of the second reagent.

(4) The absorbance change per minute (ΔAbs. / Min) was calculated from the absorbance measured in (3) above.

(5) Further, except that the imidazole-containing first reagent prepared in (1) above is used instead of the arginine-containing first reagent, the operation is performed as in the above (1) to (4), and this is also performed for 1 minute. The amount of change in absorbance per unit (ΔAbs. / Min) was calculated.

4). Measurement Results FIG. 11 shows the measurement results when using the arginine-containing first reagent as the first reagent, and FIG. 12 shows the measurement results when using the imidazole-containing first reagent as the first reagent.

  In these figures, the horizontal axis represents the calcium concentration (mg / dL) in the sample, and the vertical axis represents the amount of change in absorbance per minute (ΔAbs. / Min) obtained by measurement.

From these figures, the following can be understood.
When the arginine (basic amino acid), which is a nitrogen-containing compound, is contained in the measurement reagent, the relationship between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) [calibration curve] compared to when the reagent is not contained It became more linear and it was confirmed that the linearity of the calibration curve was improved. And it turns out that the quantitative property of a measurement is improving by this.

  In addition, when imidazole (heterocyclic compound), which is a nitrogen-containing compound, is contained in the measurement reagent, the relationship between the calcium concentration in the sample and the amount of change in absorbance (ΔAbs. / Min) [calibration] It was confirmed that the linear curve was more linear and the linearity of the calibration curve was improved. And it turns out that the quantitative property of a measurement is improving by this.

It is the graph which showed the result of having measured the calcium in a sample using bis (4-nitrophenyl) phosphoric acid, 4-nitrophenyl phenyl phosphoric acid, or 4-nitrophenyl phosphoric acid as a substrate of phospholipase D. It is the graph which showed the result of having made the measuring reagent contain cobalt ion and measuring the calcium in a sample. It is the graph which showed the result of having made the measuring reagent contain nickel ion and measuring the calcium in a sample. It is the graph which showed the result of having made a measuring reagent contain zinc ion and measuring calcium in a sample. It is the graph which showed the result of having made the measuring reagent contain citric acid which is a chelating agent, and measuring the calcium in a sample. It is the graph which showed the result of having made the measuring reagent contain oxalic acid, tartaric acid, or citric acid which are chelating agents, and measuring the calcium in a sample. It is the graph which showed the time course of the measurement reaction when BO-10TX which is a nonionic surfactant is contained in a measurement reagent. It is the graph which showed the result of having contained LMT which is anionic surfactant in a measurement reagent, and measuring the calcium in a sample. It is the graph which showed the result of having made a measurement reagent contain various surfactant and measuring the sample blank value (serum blank value). It is the graph which showed the result of having made the measuring reagent contain ammonium sulfate which is a nitrogen-containing compound, and measuring the calcium in a sample. It is the graph which showed the result of having made the measurement reagent contain the nitrogen-containing compound arginine and measuring the calcium in a sample. It is the graph which showed the result of having made the measuring reagent contain the imidazole which is a nitrogen-containing compound and measuring the calcium in a sample.

Claims (10)

At least phospholipase C and / or phospholipase D and the following general formula (I) as a substrate of phospholipase C and / or phospholipase D (wherein X1 is a chromophoric group having a nitrophenyl group, and X2 is a hydrogen atom or A reagent for measuring calcium in a sample, which comprises a compound represented by the formula: a chromophoric group having a nitrophenyl group, and X3 is a hydrogen atom.

The reagent for measuring calcium in a sample according to claim 1, comprising a substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to 0.03 to 10 mM. The calcium measurement in a sample according to claim 1 or 2, comprising at least one substance selected from the group consisting of metal ions, chelating agents, amines, basic amino acids, or heterocyclic compounds. reagent. The reagent for measuring calcium in a sample according to any one of claims 1 to 3, comprising a nonionic surfactant and / or an anionic surfactant. A chromophoric group having a nitrophenyl group is a 4-nitrophenyl group, 2-chloro-4-nitro It is a phenyl group or a 2,6-dichloro-4-nitrophenyl group, The reagent for measuring calcium in a sample according to any one of claims 1 to 4. A method for measuring calcium in a sample,
a) The following general formula (I) as a substrate of the sample, phospholipase C and / or phospholipase D, and phospholipase C and / or phospholipase D (wherein X1 is a chromophoric group having a nitrophenyl group ; X2 is a chromophoric group having a hydrogen atom or a nitrophenyl group, and X3 is a hydrogen atom), and reacting to release a chromophoric group having a nitrophenyl group from the compound. ,as well as

b) A method for measuring calcium in a sample, comprising a step of colorimetrically measuring the chromophoric group having a liberated nitrophenyl group . 7. The method for measuring calcium in a sample according to claim 6 , wherein in the step a), a substance that adjusts the apparent Km value of phospholipase C and / or phospholipase D to 0.03 to 10 mM coexists. . In the step a), metal ions, chelating agents, characterized in that the coexistence of at least one substance amines are selected from the group consisting of basic amino acids, or heterocyclic compounds, according to claim 6 or claim 7 A method for measuring calcium in the sample described. The measurement of calcium in a sample according to any one of claims 6 to 8 , wherein a nonionic surfactant and / or an anionic surfactant coexists in the step a). Method. A chromophoric group having a nitrophenyl group is a 4-nitrophenyl group, 2-chloro-4-nitro It is a phenyl group or a 2,6-dichloro-4-nitrophenyl group, The method for measuring calcium in a sample according to any one of claims 6 to 9.

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