WO2011136063A1 - Procédé de mesure d'une substance spécifique, et kit de mesure d'une substance spécifique - Google Patents

Procédé de mesure d'une substance spécifique, et kit de mesure d'une substance spécifique Download PDF

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WO2011136063A1
WO2011136063A1 PCT/JP2011/059489 JP2011059489W WO2011136063A1 WO 2011136063 A1 WO2011136063 A1 WO 2011136063A1 JP 2011059489 W JP2011059489 W JP 2011059489W WO 2011136063 A1 WO2011136063 A1 WO 2011136063A1
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adp
specific substance
nad
measuring
reaction
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PCT/JP2011/059489
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Japanese (ja)
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健太 野田
善郎 佐藤
良 小島
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日東紡績株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the present invention relates to a method for measuring a specific substance in a sample and a kit for measuring a specific substance. More specifically, the present invention relates to a method for measuring a specific substance in a sample that generates ADP by an enzyme reaction using a reagent ATP, and a kit for measuring a specific substance.
  • a specific substance that generates ADP by an enzymatic reaction using reagent ATP that is, an enzyme that generates ADP from ATP or a substrate thereof.
  • specific substances include urea or urea amide lyase, creatine or creatine kinase, choline or choline kinase.
  • Patent Document 1 and Patent Document 2 various methods have been developed in this regard.
  • Non-Patent Document 1 A method for measuring ADP from a decrease in NADH is known (Non-Patent Document 1). However, since this method is a measurement method in which the absorbance decreases as the amount of ADP in the sample increases, the upper limit value of ADP that can be measured may be limited by NADH in the reagent.
  • phosphoenolpyruvate and pyruvate kinase are allowed to act on ADP in a sample, and then the generated pyruvate is allowed to act with pyruvate oxidase or the like.
  • Patent Document 3 There is known a method for measuring the above (Patent Document 3).
  • this method is easily affected by reducing substances such as uric acid and ascorbic acid in the sample, and coloring substances such as bilirubin and hemoglobin, which may affect the measured value.
  • An object of the present invention is to measure glucose-6 produced by reacting glucose, ADP-dependent hexokinase, and metal ions with the generated ADP when measuring a specific substance in a sample that generates ADP by an enzymatic reaction using ATP.
  • An object of the present invention is to provide a stable and inexpensive kit for measuring a specific substance to be used.
  • the present inventors apply the above-mentioned method that is capable of accurately measuring ADP by subjecting a specific substance to an enzymatic reaction to generate ADP, and then allowing ADP-dependent hexokinase or the like to act on the generated ADP.
  • thio-NAD (P) and NAD (P) as coenzymes in combination with specific substances such as high concentrations of urea.
  • P thio-NAD
  • P NAD
  • a method for measuring a specific substance in a sample that generates ADP by an enzymatic reaction using ATP i) a step of allowing glucose, ADP-dependent hexokinase, and a metal ion to act on ADP present as an impurity in ATP as a reagent component; ii) a step of allowing glucose-6-phosphate dehydrogenase and NADs to act on the resulting glucose-6-phosphate, and iii) eliminating the NADHs by causing pyruvate and lactate dehydrogenase to act on the generated NADHs.
  • ADP removal reaction in which ADP in ATP as a reagent component is removed by the step of removing; and then iv) in the sample using ATP as the reagent component from which ADP has been removed by the ADP removal reaction comprising steps i) to iii) above A step of subjecting the specific substance to an enzymatic reaction to generate ADP, v) allowing glucose, ADP-dependent hexokinase, and metal ions to act on the ADP produced; vi) a step of allowing glucose-6-phosphate dehydrogenase and NAD (P) to act on the produced glucose-6-phosphate, and vii) a step of measuring the produced NAD (P) H in the sample.
  • a method for measuring a specific substance comprising a specific substance measurement reaction for measuring a specific substance; [2].
  • the specific substance measurement reaction comprising the steps of iv) to vii) under the conditions in which the lactate dehydrogenase reaction of step iii) in the ADP removal reaction does not proceed
  • the reaction solution in any step of vi) to vii) in the specific substance measurement reaction contains a lactate dehydrogenase inhibitor from the above [2] to [ 4]
  • step vi) the step of using NAD (P) and thio-NAD (P) coexisting as NAD (P) s and measuring NAD (P) Hs in step vii)
  • the method for measuring a specific substance according to any one of the above [1] to [6], which is carried out by measuring an increase in absorbance around a wavelength of 405 nm derived from NAD (P) H; [8].
  • step iv) in which the specific substance is urea and the specific substance is subjected to an enzymatic reaction to generate ADP, ATP, magnesium ion, potassium ion, hydrogen carbonate ion, and urea amide lyase are allowed to act on urea in the sample.
  • a kit for measuring a specific substance which includes a reagent component that is essential for the step of generating ADP and is other than ATP, independently of other reagent components; [10].
  • the specific substance is urea, essential for the step of subjecting the specific substance to an enzymatic reaction to generate ADP, and the reagent components other than ATP are magnesium ion, potassium ion, bicarbonate ion, and urea amidolyase, [9] a kit according to [9]; and [11].
  • ADP present in ATP as a reagent is converted into ADP-dependent hexokinase reaction, glucose-6-phosphate dehydrogenation.
  • ADP is measured, so that the measurement blank is low and the slope of the calibration curve is small, so that a wide range of ADP concentrations can be accurately measured.
  • thio-NAD (P) and NAD (P) are used in combination as a coenzyme for a specific substance measurement reaction, it becomes easy to measure a high concentration of a specific substance.
  • FIG. 1 shows a decrease in blank value when urea is measured under the conditions of Example 1-7 using thio-NADP and NADP but not using an inhibitor (oxalic acid) in the present invention.
  • FIG. 2 shows a decrease in the blank value when urea is measured under the conditions of Comparative Example 1-7 in which lactate dehydrogenase is not used.
  • FIG. 3 shows the results of a calibration curve when urea is measured under the conditions of Example 1-7 using thio-NADP and NADP and not using an inhibitor (oxalic acid) in the present invention.
  • FIG. 4 shows a decrease in blank value when urea is measured under the conditions of Examples 8-14 using thio-NAD and NAD and further using an inhibitor (oxalic acid) in the present invention.
  • FIG. 5 shows a decrease in the blank value when urea is measured under the conditions of Comparative Examples 8-14 without using lactate dehydrogenase.
  • FIG. 6 shows the result of a calibration curve when urea is measured under the conditions of Examples 8-14 using thio-NAD and NAD and further using an inhibitor (oxalic acid) in the present invention.
  • the sample used in the present invention is a sample containing a specific substance that converts ATP to ADP by an enzymatic reaction, and is preferably not particularly limited as long as it is liquid, but biological samples such as serum, plasma, urine, etc.
  • the model sample is preferred.
  • the specific substance is not particularly limited as long as it is a substance that generates ADP by an enzymatic reaction using ATP, that is, an enzyme that generates ADP from ATP or a substrate thereof. Examples of such specific substances include urea or urea amide lyase, creatine or creatine kinase, choline or choline kinase.
  • urea amide lyase reaction in which urea, ATP, magnesium ion, potassium ion, hydrogen carbonate ion and urea amide lyase act can be used as the specific substance enzyme reaction.
  • the reaction formula is shown below.
  • a creatine kinase reaction in which creatine, ATP, magnesium ions and creatine kinase act can be used as the specific substance enzyme reaction.
  • the reaction formula is shown below.
  • a choline kinase reaction in which choline, ATP, magnesium ion, or choline kinase acts can be used as the specific substance enzyme reaction.
  • the reaction formula is shown below.
  • ADP refers to adenosine diphosphate.
  • ATP refers to adenosine triphosphate (Adenosine triphosphate).
  • NAD means, for example, any of NAD, NADP, thio-NAD, or thio-NADP, and NAD means, for example, any of NAD, thio-NAD, and NADPs Means, for example, NADP or thio-NADP, but it is known that they have a structure similar to those that function as a coenzyme used in the dehydrogenase reaction, for example, the ring skeleton is the same and has a function as a coenzyme. It may be a compound.
  • NAD means nicotinamide adenine dinucleotide
  • NADP means nicotinamide adenine dinucleotide phosphate
  • thio NAD means thionicotinamide adenine dinucleotide
  • thio NADP means thionicotinamide adenine Means dinucleotide phosphate.
  • NAD (P) H is a corresponding reduced form of NAD (P).
  • NAD (P) is NAD, NADP, thio-NAD, or thio-NADP, , NADPH, thio-NADH, thio-NADPH.
  • NAD means NAD or NADP
  • NAD (P) H means a corresponding reduced form of NAD (P)
  • thio NAD means thio NAD or thio NADP
  • thio NAD (P) H means a corresponding reduced form of thio NAD (P)
  • thio NAD (P) is Thio NAD and thio NADP mean thio NADH and thio NADPH, respectively.
  • the method for measuring a specific substance in a sample includes an ADP removal reaction for removing ADP in ATP as a reagent component comprising steps i) to iii) below, and steps iv) to vii) below.
  • a specific substance measurement reaction for measuring a specific substance in a sample consisting of
  • the ADP removal reaction comprising the steps i) to iii) and the specific substance measurement reaction comprising the steps iv) to vii) are preferably carried out in one pot, that is, all at once.
  • ADP removal reaction i) Step of allowing glucose, ADP-dependent hexokinase, and metal ion to act on ADP present as an impurity in ATP as a reagent component
  • this reaction is ADP-dependent
  • ADP is caused to react with glucose, ADP-dependent hexokinase, and metal ions to produce glucose-6-phosphate (G6P).
  • G6P glucose-6-phosphate
  • ADP is ADP present as an impurity in ATP as a reagent component.
  • magnesium ion As the metal ion, magnesium ion, cobalt ion, manganese ion and the like are preferable.
  • the glucose-6-phosphate (G6P) produced in step i) is the enzyme Subject to reaction.
  • Glucose-6-phosphate dehydrogenase in this specification, a reaction involving glucose-6-phosphate dehydrogenase is also referred to as glucose-6-phosphate dehydrogenase reaction
  • G6PDH glucose-6-phosphate dehydrogenase reaction
  • a lactate dehydrogenase reaction is used.
  • This lactate dehydrogenase reaction proceeds when NADHs are used as coenzymes, and does not proceed when NADPHs are used. It is necessary that the coenzyme used in) is NADs and that NADHs are produced so that the lactate dehydrogenase reaction in the next step iii) proceeds.
  • step vii) A step of removing NADHs by allowing pyruvate and lactate dehydrogenase to act on the produced NADHs
  • step vii) the NAD (P) Hs generated in step vi) are measured in step vii). Since NADHs produced in ii) cause an increase in the blank, in this step iii), NADHs produced in step ii) are subjected to NADH by a lactate dehydrogenation reaction in which pyruvate and lactate dehydrogenase act. Class is converted to NAD class and deleted.
  • ATP is obtained as a reagent component from which ADP has been removed by the ADP removal reaction comprising the steps i) to iii) described above, and the following specific substance measurement reaction is performed using this ATP.
  • Specific substance measurement reaction iv) A step of generating ADP by subjecting a specific substance in a sample to an enzymatic reaction using ATP as a reagent component from which ADP has been removed by the ADP removal reaction comprising the steps i) to iii) in this step iv)
  • the specific substance enzyme reaction using a reagent component for advancing the specific substance enzyme reaction removes ADP by the specific substance in the sample to be measured and the ADP removal reaction comprising the steps i) to iii) above.
  • ADP is generated from ATP as a reagent component.
  • urea or urea amide lyase as a specific substance, as is apparent from the above-mentioned urea amide lyase reaction, as a reagent component for the specific substance enzyme reaction, urea amide lyase or urea, ATP, magnesium ion, potassium ion Hydrogen carbonate ions are used.
  • the reagent components for the specific substance enzyme reaction can be similarly determined from the creatine kinase reaction and the choline kinase reaction.
  • Step of allowing glucose, ADP-dependent hexokinase, and metal ion to act on ADP to be generated This step is a step of subjecting ADP generated in step iv) to an ADP-dependent hexokinase reaction.
  • magnesium ion As the metal ion, magnesium ion, cobalt ion, manganese ion and the like are preferable.
  • step v) Step of allowing glucose-6-phosphate dehydrogenase and NAD (P) to act on the glucose-6-phosphate to be produced
  • glucose-6-phosphate produced by step v) Glucose-6-phosphate dehydrogenase (G6PDH) is allowed to act on acid (G6P) to perform glucose-6-phosphate dehydrogenase, and NADHs together with gluconolactone-6-phosphate (6-PGL) Produces.
  • G6PDH Glucose-6-phosphate dehydrogenase
  • G6P Glucose-6-phosphate dehydrogenase
  • NADHs gluconolactone-6-phosphate
  • 6-PGL gluconolactone-6-phosphate
  • NAD (P) H has almost no absorbance in NAD (P) s and uses a wavelength in which NAD (P) H has absorbance
  • NAD (P) Hs Measure the increase in absorbance from origin.
  • the concentration of the specific substance can be measured from the amount of increase in absorbance derived from the NAD (P) H, as compared with a calibration curve prepared in advance.
  • step vi) when NAD (P) is used as the coenzyme NAD (P), ADP is determined from the increase in absorbance at around 340 nm derived from the generated NAD (P) H, for example, at a wavelength of 330 to 350 nm. Can be measured.
  • the absorbance at around 405 nm, for example, at a wavelength of 390 to 415 nm, derived from the generated thio-NAD (P) H ADP can be measured from the amount of increase.
  • the use of thio-NAD (P) in the present invention is preferable in that it can be measured using a visible spectrophotometer that is inexpensive and easily available, and the measurement area of a specific substance can be remarkably improved.
  • step vi) glucose-6-phosphate dehydrogenase reaction is performed in the presence of thio-NAD (P) and NAD (P) as coenzymes, and thio-NAD It is particularly preferred to measure ADP by generating (P) H and NAD (P) H and measuring the increase in absorbance around a wavelength of 405 nm derived from the generated thio-NAD (P) H.
  • the amount of NAD (P) used is not particularly limited, but is preferably 0.1 to 30 times the number of thioNAD (P) used in order to adjust the slope of the calibration curve appropriately, A mole number of ⁇ 25 times is more preferred.
  • step iii) after removing ADP in the reagent component by the ADP removal reaction comprising steps i) to iii), iv) to vii) under the conditions where the lactate dehydrogenase reaction of step iii) does not proceed. It is preferable to carry out a specific substance measurement reaction consisting of these steps.
  • the lactate dehydrogenase reaction in step iii) proceeds in the specific substance measurement reaction
  • the reverse reaction in which the NADHs produced in step vi) are converted to NADs is an error in measuring the NAD (P) Hs in step vii) It is because it becomes the cause.
  • the NAD (P) s used in step vi) are: NADPs are preferred.
  • glucose-6-phosphate dehydrogenase preferably has higher substrate affinity for NADPs than for NADs.
  • the glucose-6-phosphate dehydrogenase preferably has a Km for NADPs of 0.01 to 0.5 times the Km for NADs.
  • the reaction solution It is also preferable to inhibit the lactate dehydrogenase reaction by allowing a lactate dehydrogenase inhibitor to be present.
  • the lactate dehydrogenase inhibitor include oxalic acid, oxamic acid, and salts thereof, and oxalic acid is preferable among them.
  • a first reagent for advancing the ADP removal reaction consisting of steps i) to iii), and a specific substance measurement reaction consisting of steps iv) to vii) It is preferable to use a specific substance measurement kit composed of the second reagent.
  • the first reagent contains glucose, ADP-dependent hexokinase, metal ion, glucose-6-phosphate dehydrogenase, NADs, pyruvate, lactate dehydrogenase, and ATP as essential components. Is preferred.
  • the first reagent containing ATP after mixing essential components, is removed from ATP as a pretreatment, for example, at room temperature to 37 ° C., preferably at room temperature for 1 hour or longer. It is preferable.
  • the steps i) to iii) can be achieved in one pot, that is, at once.
  • the second reagent contains NAD (P) as an essential component as a component for advancing the specific substance measurement reaction consisting of steps iv) to vii), but the components contained in the first reagent are omitted. May be.
  • the first reagent or the second reagent is required for the step of generating ADP by subjecting a specific substance to an enzyme reaction, and the reagent components other than ATP must be included independently, and at least the essential components It is preferable that a part or all of the components are contained in the second reagent.
  • the specific substance is urea
  • reagent components that are essential for the step of generating ADP by subjecting the specific substance to an enzymatic reaction and are other than ATP are magnesium ion, potassium ion, bicarbonate ion, and urea amide lyase. Yes, these must be independently contained in the first reagent or the second reagent.
  • it is preferable that at least a part or all of the essential components are contained in the second reagent.
  • the components of the kit for measuring a specific substance of the present invention are, for example, NADs, NAD (P) s, metal ions, glucose-6-phosphate dehydrogenase and the like described in the method for measuring a specific substance of the present invention.
  • NADs NAD (P) s
  • metal ions glucose-6-phosphate dehydrogenase and the like described in the method for measuring a specific substance of the present invention.
  • a thing can be appropriately selected and used according to the effect.
  • the second reagent preferably contains a lactate dehydrogenase inhibitor.
  • a surfactant and a buffering agent may be appropriately added to the first reagent or the second reagent.
  • a specific substance can be measured by this kit using an automatic analyzer or a visible part measuring device.
  • the first reagent that has undergone steps i) to iii) is mixed at 20 to 40 ° C., preferably 37 ° C., and allowed to stand for 3 to 10 minutes, preferably 5 minutes.
  • a sample and a second reagent are added thereto, and a specific substance measurement reaction including the steps iv) to vii) is performed in one pot, that is, at once, and an increase in the generated NAD (P) H is measured at an appropriate wavelength.
  • P NAD
  • Example 1-7 and Comparative Example 1-7 Measurement of urea according to the invention using thio-NADP and NADP and no inhibitor (oxalic acid) METHOD urea amide lyase, ADP-dependent hexokinase, and glucose-6-phosphate dehydrogenase, thio NADP, in urea nitrogen measurement system using NADP, from ADP contained in the ATP in the reagent, ADP-dependent hexokinase , And the color development of NADH caused by the reaction of glucose-6-phosphate dehydrogenase was previously prevented by oxidizing NADH to NAD and eliminating it by a reaction using lactate dehydrogenase in the reagent. By this method, the blank value and the measurable concentration range of urea were examined. Furthermore, it compared with the comparative example measured without oxidizing and eliminating NADH into NAD without using lactate dehydrogenase.
  • First reagent As the first reagent, one having the following composition was used. 100 mM BES (N, N-bis (2-hydroxyethyl-2- Aminoethanesulfonic acid) pH 7.5 10 mM D-glucose 10 mM potassium bicarbonate 5 mM ATP ⁇ 2Na (including various concentrations of ADP) 10 mM pyruvate 5 mM NAD 0.1% Surfactant 6KU / L Hexokinase 2KU / L Ureaamide lyase 2KU / L Glucose-6-phosphate dehydrogenase 200U / L Lactate dehydrogenase
  • Second reagent As the second reagent, one having the following composition was used. 100 mM BES (N, N-bis (2-hydroxyethyl-2- Aminoethanesulfonic acid) pH 7.5 10mM magnesium acetate tetrahydrate 5mM NADP 1 mM Thio NADP 0.1% surfactant
  • reagent composition In this example, as shown in Table 1, a reagent in which ADP is present in the ATP of the reagent component was used.
  • the first reagent used in this example was allowed to stand for 1 hour or longer so that NADH generated from ADP became NAD by lactate dehydrogenase, and sufficient time was allowed to remove ADP in ATP.
  • the coenzyme used in the ADP measurement system (step vi in the specific substance measurement reaction) is not thioNAD that reacts with LDH but thio that does not react with LDH. NADP was used.
  • NADP is not added as a coenzyme
  • thio-NADP alone has a measurement blank of 2 times or more and has a high slope and a measurement concentration range of 50 mg / dL or less. Therefore, use thio-NADP and NADP together. I made it.
  • the first reagent used in Comparative Examples 1 to 7 did not add LDH and used a composition that does not cause an ADP elimination reaction.
  • a reagent composition was used in which the final concentration ratio of NADP to thio-NADP was 5: 1.
  • the measurement was performed as follows. Using a visible measurement device (absorbance confidence range of 2.5 or less), 3.0 ⁇ L of the sample and 100 ⁇ L of the first reagent were mixed at 37 ° C. for 5 minutes, and then 100 ⁇ L of the second reagent was added and reacted at the same temperature for 5 minutes. . The absorbance after the color reaction was measured at a wavelength of 405 nm by the one-point end method of the model.
  • the calibration curve of Comparative Comparative Example 1-7 b) Linearity Figure 2 shows a calibration curve of Example 1-7 in FIG.
  • FIG. 2 in the composition of Comparative Example 1-7, when a sample having a urea nitrogen concentration of 200 mg / dL was measured, the obtained absorbance exceeded 2.5, which is the upper limit of absorbance of the measuring instrument, and measurement was impossible.
  • FIG. 3 in Example 1-7, the absorbance obtained by measuring a sample having a urea nitrogen concentration of 200 mg / dL does not exceed 2.5, and the measurable range of urea is expanded by the present invention. It has been found.
  • Example 8-14 and Comparative Example 8-14 Measurement of urea according to the invention using thio-NAD and NAD and further using an inhibitor (oxalic acid) Method Aurea lyase, ADP-dependent hexokinase, and ADP-dependent hexokinase from ADP contained in ATP in the reagent in a urea nitrogen measurement system using glucose-6-phosphate dehydrogenase, thio-NAD, NAD , And the color development of NADH caused by the reaction of glucose-6-phosphate dehydrogenase was previously prevented by oxidizing NADH to NAD and eliminating it by a reaction using lactate dehydrogenase in the reagent.
  • an inhibitor oxalic acid
  • First reagent As the first reagent, one having the following composition was used. 100 mM BES (N, N-bis (2-hydroxyethyl-2- Aminoethanesulfonic acid) pH 7.5 10 mM D-glucose 10 mM potassium bicarbonate 5 mM ATP ⁇ 2Na (including various concentrations of ADP) 10 mM pyruvate 5 mM NAD 0.1% Surfactant 6KU / L Hexokinase 2KU / L Ureaamide lyase 2KU / L Glucose-6-phosphate dehydrogenase 200U / L Lactate dehydrogenase
  • Second reagent As the second reagent, one having the following composition was used. 100 mM BES (N, N-bis (2-hydroxyethyl-2- Aminoethanesulfonic acid) pH 7.5 10 mM Magnesium acetate tetrahydrate 100 mM Potassium oxalate monohydrate 1 mM Thio NAD 0.1% surfactant
  • reagent composition In this example, as shown in Table 3, a reagent in which ADP is present in the ATP of the reagent component was used. The first reagent used in this example was used with sufficient time so that NADH generated from ADP becomes NAD by lactate dehydrogenase (LDH). Further, oxalic acid, which is an inhibitor against LDH, was added to the second reagent composition so that NADH generated from urea nitrogen in the sample was not erased by LDH. As shown in Table 3, the first reagent used in the comparative example did not add LDH and used a composition that did not erase ADP.
  • LDH lactate dehydrogenase
  • the measurement blank is twice or more, the slope is high, and the measurement concentration range is 50 mg / dl or less. Therefore, with the aim of expanding the measurable range of the measurement system, the end of NAD and thio NAD A reagent composition having a concentration ratio of 5: 1 was used.
  • FIG. 5 shows the calibration curve of Example 8-14 in FIG.
  • Comparative Example 8-14 when a sample having a urea nitrogen concentration of 200 mg / dL was measured, the obtained absorbance exceeded 2.5, which is the upper limit of absorbance of the measuring instrument, and measurement was impossible.
  • FIG. 6 in Examples 8-14, the absorbance obtained even when measuring a sample having a urea nitrogen concentration of 200 mg / dL does not exceed 2.5, and the measurement range of urea is expanded by the present invention. There was found.
  • ADP present in ATP as a reagent is converted into ADP-dependent hexokinase reaction, glucose-6-phosphate dehydrogenation.
  • ADP is measured, so that the measurement blank is low and the slope of the calibration curve is small, so that a wide range of ADP concentrations can be accurately measured.
  • thio-NAD (P) and NAD (P) are used in combination as a coenzyme in the measurement system reaction, it becomes easy to measure a specific substance at a high concentration.
  • a wide range of concentrations of specific substances in a sample such as urea or urea amide lyase, creatine or creatine kinase, choline or choline kinase can be accurately and efficiently measured.
  • the kit used is extremely useful for diagnosis of various diseases.

Abstract

La présente invention concerne un procédé de mesure d'une substance spécifique dans un échantillon, dans lequel de l'ADP est générée par une réaction enzymatique utilisant de l'ATP. Le procédé comprend les étapes consistant à : i) faire réagir l'ADP qui est présente sous la forme d'une impureté dans l'ATP qui sert de réactif avec du glucose, une hexokinase dépendante de l'ADP et un ion métallique ; ii) faire réagir le glucose-6-phosphate qui est produit dans l'étape i) avec une glucose-6-phosphate déshydrogénase et un composant NAD ; iii) faire réagir un composant NADH produit dans l'étape ii) avec de l'acide pyruvique et de la lactate déshydrogénase pour piéger le composant NADH, éliminant ainsi l'ADP dans l'ATP qui sert de réactif (les étapes i) à iii) constituent une réaction d'élimination de l'ADP) ; iv) soumettre une substance spécifique dans un échantillon à une réaction enzymatique en utilisant de l'ATP qui sert de réactif et duquel l'ADP a été éliminée par la réaction d'élimination de l'ADP qui comprend les étapes i) à iii), générant ainsi de l'ADP ; v) faire réagir l'ADP ainsi produite avec du glucose, une hexokinase dépendante de l'ADP et un ion métallique ; vi) faire réagir le glucose-6-phosphate qui est produit dans l'étape v) avec une glucose-6-phosphate déshydrogénase et un composant NAD(P) ; et vii) mesurer le composant NAD(P)H produit dans l'étape vi). Ainsi, le procédé de mesure de la substance spécifique implique une réaction de mesure de substance destinée à mesurer la substance spécifique dans l'échantillon. Le procédé permet la mesure d'une substance spécifique qui est présente dans un échantillon à une concentration qui tombe dans une vaste plage de concentrations correctement et avec une grande efficacité.
PCT/JP2011/059489 2010-04-30 2011-04-18 Procédé de mesure d'une substance spécifique, et kit de mesure d'une substance spécifique WO2011136063A1 (fr)

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US20210222227A1 (en) * 2018-10-19 2021-07-22 Sysmex Corporation Enzymatic measurement method and reagent for enzymatic measurement

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