JP6417517B2 - Reagent for measuring enzyme activity in biological sample avoiding influence of hemoglobin and measuring method - Google Patents

Description

The present invention relates to a reagent and method for measuring enzyme activity in a biological sample, and avoids the influence of hemoglobin in the measurement of enzyme activity.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

  Measuring enzyme activity in biological samples such as blood and observing the fluctuations are indispensable for diagnosis, treatment, early detection and prevention of diseases, and are widely practiced. In order to measure the enzyme activity in this biological sample, 4-nitrophenol, 2-chloro-4-nitrophenol, 4-nitro produced by reacting these enzymes with a substrate having a nitrophenyl group or a nitroaniline group or the like. A rate assay is generally used to determine the rate of increase in absorbance over time at a wavelength of 400 to 500 nm of a substance having an absorption maximum at 400 to 500 nm, such as aniline or 3-carboxy-4-nitroaniline, and to determine enzyme activity from the measurement results. Is.

  By the way, in such a method, if hemoglobin generated by hemolysis is contained in the sample, it is decomposed during measurement, causing a decrease in absorbance over time in the measurement wavelength range, and measuring enzyme activity. There is a problem that the rate of increase in absorbance in the wavelength region is reduced, a negative error occurs, and an accurate measurement value cannot be obtained.

  Therefore, in order to prevent the hemoglobin produced by hemolysis from being decomposed during the measurement, a method of containing thiourea in the enzyme activity measurement reagent (see, for example, Patent Document 1), pyridines, imidazoles, histamines. (For example, refer patent document 2) etc. were proposed, but it was not necessarily complete.

Japanese Patent Publication No. 6-12998 Japanese Examined Patent Publication No. 3-56425

  As described above, the conventional enzyme activity measuring reagent and enzyme activity measuring method in a biological sample are affected by hemoglobin in the hemolyzed sample, resulting in a negative error in the measured value. For this reason, for example, in the clinical examination in the medical field, when trying to use the measured value of the enzyme activity in the biological sample for diagnosis, there is a possibility that the diagnosis of a disease or the like may be mistaken.

Therefore, an object of the present invention is to provide a measuring reagent and a measuring method that can accurately measure the enzyme activity in a sample, even if the sample is hemolyzed, avoiding the influence of hemoglobin generated by hemolysis. It is.
Moreover, the subject of this invention is providing the method of avoiding the influence of hemoglobin, and providing the influence avoidance agent of hemoglobin.

  As a result of intensive studies aimed at solving the above problems, the present inventors have found for the first time that the above problems can be solved when zinc ions are present in the enzyme activity measurement reagent in the range of 8 to 1200 ppb. It came to complete.

That is, the present invention provides the following inventions.
(1) A reagent for measuring alkaline phosphatase activity in a biological sample containing zinc ions at a concentration of 8 to 1200 ppb and using 2-ethylaminoethanol as a buffer solution.
(2) The alkaline phosphatase activity measuring reagent in the biological sample according to (1) above, wherein the alkaline phosphatase activity measuring reagent measures absorbance in a wavelength range of 400 to 500 nm.
(3) The alkaline phosphatase activity measurement reagent is composed of two reagents, a first reagent and a second reagent, and the zinc ion is contained in at least one of the first reagent and the second reagent (1) ) Or the reagent for measuring alkaline phosphatase activity in a biological sample according to (2).
(4) The reagent for measuring alkaline phosphatase activity in a biological sample according to any one of (1) to (3) above, wherein zinc ions are contained as an influence avoiding agent for hemoglobin.
(5) An alkaline phosphatase activity measuring reagent containing zinc ions at a concentration of 8 to 1200 ppb and using 2-ethylaminoethanol as a buffer is mixed with a biological sample, and an enzyme reaction is performed in the presence of zinc ions. A method for measuring alkaline phosphatase activity in a biological sample, wherein the absorbance in a wavelength region of 400 to 500 nm is measured.
(6) The alkaline phosphatase activity measuring reagent is composed of two reagents, a first reagent and a second reagent, and the zinc ion is contained in at least either the first reagent or the second reagent (5) The method for measuring alkaline phosphatase activity in a biological sample according to (1).
(7) The method for measuring alkaline phosphatase activity in a biological sample according to the above (5) or (6), wherein zinc ions are contained as an influence avoiding agent for hemoglobin.
( 8 ) An agent for avoiding the influence of hemoglobin at the time of measuring alkaline phosphatase activity in a biological sample using 2-ethylaminoethanol as a buffer, comprising zinc ions having a concentration of 8 to 1200 ppb.

  According to the present invention, in the enzyme activity measurement reagent and measurement method in a biological sample, the influence of hemoglobin is avoided by including a trace amount of zinc ions at a concentration of 8 to 1200 ppb in the enzyme activity measurement reagent, and the measured value It is possible to prevent the occurrence of an error.

  That is, the reagent for measuring enzyme activity in the biological sample of the present invention, the measuring method, the method for avoiding the influence of hemoglobin, and the agent for avoiding the influence of hemoglobin are accurate without being affected by the hemoglobin produced by the hemolytic action. A measured value can be obtained.

  Thereby, the enzyme activity measuring reagent, the measuring method, the method for avoiding the influence of hemoglobin, and the hemoglobin influence avoiding agent in the biological sample of the present invention can prevent misdiagnosis in a medical field, for example. Is.

  Hereinafter, the present invention will be described in detail. However, the following embodiments are examples for explaining the present invention, and the present invention is not limited to these embodiments. Moreover, this invention can be implemented with various forms, unless it deviates from the summary.

[1] Zinc ion In the present invention, the enzyme activity measuring reagent contains zinc ion. Any zinc ion may be included in the measurement reagent as long as it is a zinc ion. For example, a compound containing zinc ions can be used.
Specific examples include compounds composed of zinc ions and acid groups, hydroxides of zinc ions, and salts of zinc ions.
More specifically, zinc salts such as zinc sulfate, zinc nitrate, zinc acetate, zinc lactate, or zinc chloride can be used.

  In the present invention, in order to avoid a negative error in the hemolyzed sample, the concentration of zinc ions contained in the enzyme activity measurement reagent may be 8 to 1200 ppb, preferably 8 to 1000 ppb, more preferably 8 to 800 ppb, particularly preferably 8 to 600 ppb.

In the enzyme activity measurement reagent of the present invention, when the measurement reagent consists of one reagent, the concentration of zinc ions contained in the measurement reagent may be in the above range, and the measurement reagent may be the first reagent and When the second reagent is composed of two reagents, when the first reagent and the second reagent are mixed at the ratio of the respective addition amounts when the enzyme activity in the biological sample is measured, the measurement reagent after mixing is measured. Zinc ions may be contained in either the first reagent or the second reagent so that the concentration of the zinc ions in the range falls within the above range.
Moreover, as long as the concentration of zinc ions in the measurement reagent after mixing falls within the above concentration range, zinc ions may be contained in both the first reagent and the second reagent.
The same applies when the measurement reagent is composed of three or more reagents.

In the present invention, zinc ions are present in the enzyme activity measurement reagent. At least in the measurement reaction in which the enzyme reaction between the biological sample and the reagent component is performed, the presence of zinc ions causes hemoglobin produced by hemolysis. The influence can be suppressed.
Accordingly, in the enzyme activity measurement reagent of the present invention, zinc ions may be contained in at least one of the first reagent and the second reagent, and may be contained in both the first reagent and the second reagent. In addition, in order to suppress the influence of hemoglobin generated by hemolysis, it is preferable that zinc ions are contained in the first reagent.

  In the enzyme activity measuring reagent of the present invention, the first reagent and the second reagent are mixed in the ratio of the addition amount at the time of measurement, and the zinc ion concentration in the measuring reagent and the first reagent and the second reagent are included. The formula of the relationship of the zinc ion concentration is shown in the following formula 1.

  In Equation 1, CM is the zinc ion concentration (ppb) in the measurement reagent after mixing, C1 is the zinc ion concentration (ppb) contained in the first reagent, and C2 is the zinc ion concentration contained in the second reagent. The concentration (ppb), V1 represents the ratio of the added amount of the first reagent at the time of measurement, and V2 represents the ratio of the added amount of the second reagent at the time of measurement.

When the ratio (V1, V2) of the addition amount of the first reagent and the second reagent at the time of measurement is determined, the zinc ion concentration (CM) in the measurement reagent after mixing is 8 to 1200 ppb (preferably 8 to 1000 ppb). More preferably, the concentration of zinc ions to be contained in the first reagent and the second reagent may be determined according to Formula 1 so that the specific value is within the range of 8 to 800 ppb, particularly preferably 8 to 600 ppb.
The same applies when the measurement reagent is composed of three or more reagents.

  By the way, in this invention, the density | concentration in an enzyme activity measuring reagent, or the density | concentration in the measuring reagent after mixing the reagent which comprises measuring reagents, such as a 1st reagent and a 2nd reagent, is set to the density | concentration of zinc ion 8-8. It is defined as 1200 ppb (preferably 8-1000 ppb, more preferably 8-800 ppb, particularly preferably 8-600 ppb). This is the concentration after mixing the enzyme activity measurement reagent and the biological sample, or the first reagent, There is no problem even if it is replaced with the concentration after mixing the reagent constituting the measurement reagent such as two reagents and the biological sample, that is, the concentration in the final reaction solution of the enzyme activity measurement reaction.

  The ratio of the amount of sample added to the amount of the reagent for measuring the enzyme activity is very small when the amount of sample added is divided by the amount of the reagent for measuring enzyme activity. Therefore, it is usually within the range of 1 to 5%, and there is no great difference whether or not the amount of sample added is considered.

[2] pH when measuring enzyme activity
In the present invention, the pH at the time of enzyme activity measurement is not particularly limited, but may be in the pH range where the reaction at the time of enzyme activity measurement proceeds. In addition, it is preferable that pH at the time of this measurement reaction exists in the optimal pH range of the said reaction, or its vicinity.
For example, enzymes such as alkaline phosphatase (ALP), leucine aminopeptidase (LAP), γ-glutamyltransferase (γ-GT), cholinesterase (ChE) and the like have an optimum pH range in the range of pH 7-14. It is preferable to adjust the pH at the time of activity measurement to around pH 7 to 14.

  Moreover, 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 buffers that can be used include phosphoric acid, tris (hydroxymethyl) aminomethane, glycylglycine, monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N- (2-acetamido) iminodiacetic acid (ADA), 2-amino-2-methyl-1-propanol (AMP), N, N-bis ( 2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N, N-bis (2-hydroxyethyl) glycine (Bicine), 2,2-bis (hydroxyethyl)-(iminotris)-(hydroxymethyl) Methane (Bis-Tris), 1,3-bistrishydroxymethylmethylamino Pan (Bis-Trispropane), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-hydroxy-3-aminoethanesulfonic acid (CAPS), N-cyclohexyl-2- Aminoethanesulfonic acid (CHES), 3- [N, N-bis (hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 2-ethylaminoethanol (EAE), 2- [4- (2- Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] -propanesulfonic acid monohydrate (HEPPSO), N-methyl- D-glucamine (MEG), 2-morpholinoethanesulfonic acid ( ES), 3-morpholinopropane sulfonic acid (MOPS), 2-hydroxy-3-morpholinopropane sulfonic acid (MOPSO), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), piperazine-1,4 -Bis (2-hydroxy-3-propanesulfonic acid) dehydrate (POPSO), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-tris (hydroxymethyl) methyl-2-hydroxy -3-aminopropanesulfonic acid (TAPSO), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), or N- [tris (hydroxymethyl) methyl] glycine (Tricine), or a salt thereof And each buffering agent.

  In the present invention, the buffer solution is 2-amino-2-methyl-1-propanol (AMP), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-3- Aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-hydroxy-3-aminoethanesulfonic acid (CAPS), 2-ethylaminoethanol (EAE), N-methyl-D-glucamine (MEG), monoethanolamine ( An alkaline buffer solution such as MEA), diethanolamine (DEA), and triethanolamine (TEA) is preferable.

  In the present invention, the buffer solution is particularly 2-amino-2-methyl-1-propanol (AMP), 2-ethylaminoethanol (EAE), N-methyl-D-glucamine (MEG), diethanolamine (DEA). It is preferable that it is a buffer solution used for activity measurement of alkaline phosphatase (ALP).

[3] Biological sample In the present invention, the biological sample is intended to measure enzyme activity in a biological sample, and is not particularly limited as long as it is such.
Such biological samples include, for example, human or animal blood, serum, plasma, urine, stool, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, brain and other organs, hair, skin, nails, Examples include tissues such as muscles or nerves and extracts of cells.

[4] Enzyme in biological sample In the present invention, an enzyme that measures enzyme activity is one that can measure enzyme activity by measuring absorbance in the wavelength range of 400 to 500 nm.
In other words, a substance produced by catalyzing the reaction of the enzyme whose activity is to be measured or a compound derived from this product having absorption in the wavelength range of 400 to 500 nm may be used as a substrate. It can be an enzyme.

  Examples of such enzymes include alkaline phosphatase (ALP), allylamidase (AA), α-amylase (AMY), cholinesterase (ChE), γ-glutamyltransferase (γ-GT), leucine aminopeptidase (LAP), Examples thereof include L-cystine aminotranspeptidase (CAP).

  Moreover, in this invention, it is preferable that the enzyme which measures an enzyme activity is alkaline phosphatase (ALP).

In addition, the present invention can also be used for measuring the activity of the above-mentioned enzyme isozymes.
In this case, for example, an inhibitor such as an antibody that inhibits a specific isozyme is allowed to act on an enzyme in a biological sample and the enzyme activity is measured.

[5] Reagent for measuring enzyme activity The reagent for measuring enzyme activity in the biological sample of the present invention is characterized by containing zinc ions having a concentration of 8 to 1200 ppb. Thereby, the influence of the hemoglobin produced | generated by the hemolytic action can be avoided, and the enzyme activity in a biological sample can be measured correctly.
In addition, in the enzyme activity measuring reagent of this invention, what is necessary is just to follow the reagent which measures the enzyme activity in a conventionally well-known biological sample except containing a trace amount zinc ion of the density | concentration of 8-1200ppb.

  In addition, the enzyme activity measuring reagent of the present invention may be measured by the end point method (end point method), or may be measured by the reaction rate method (rate method). Just choose.

  The enzyme activity measuring reagent of the present invention may be a one-step method (one-reagent method) in which measurement is performed in one step, or measurement is performed in two or more steps. It may be a multi-step method (multi-reagent method) to be performed, and may be selected as appropriate.

  Further, in the reagent for measuring enzyme activity of the present invention, in addition to the above-described components, as components used for measurement reaction, stabilizers such as metal ions or metal salts containing them, chelating agents, saccharides or polymer compounds In addition, a preservative such as sodium azide, a surfactant, an excipient, an activator, or the like can be appropriately used as necessary.

  Further, in the enzyme activity measurement reagent of the present invention, the measurement may be performed by an ordinary method, or may be performed using an apparatus such as an automatic analyzer.

  Further, in the enzyme activity measuring reagent of the present invention, all or a part of the constituent reagents may be a liquid reagent.

  The reagent for measuring enzyme activity of the present invention can be sold alone or used for measuring enzyme activity in a biological sample.

Further, the enzyme activity measurement reagent of the present invention can be sold in combination with other reagents other than the above-described measurement reagents, or can be used for measurement of a measurement target substance in a sample.
Other reagents other than the above-described measurement reagents include, for example, a buffer, a sample diluent, a reagent diluent, a reagent containing a substance for performing calibration (calibration), or a substance for performing quality control. And the like.

  The enzyme activity measurement reagent of the present invention may be a measurement reagent kit comprising a plurality of constituent reagents such as the first reagent and the second reagent, or other reagents.

  In the present invention, the enzyme activity measuring reagent is preferably used for measuring the activity of alkaline phosphatase. In this case, as the configuration of the measurement reagent, for example, the first reagent contains an alkaline buffer solution, the second reagent contains an alkaline buffer solution and a substrate such as 4-nitrophenyl phosphate, and the first reagent is alkaline. Containing a buffer solution and containing a substrate such as 4-nitrophenyl phosphate in the second reagent, or containing a substrate such as 4-nitrophenyl phosphate in the first reagent and an alkaline buffer solution as the second reagent The thing of the structure which contains is mentioned.

[6] Method for measuring enzyme activity In the method for measuring enzyme activity in a biological sample of the present invention, an enzyme activity measuring reagent containing zinc ions at a concentration of 8 to 1200 ppb is mixed with the biological sample, and in the presence of a trace amount of zinc ions. The conventional enzyme activity measurement method may be followed except that the enzyme reaction is carried out and the absorbance in the wavelength region of 400 to 500 nm is measured.
In addition, the enzyme activity measurement method in the biological sample of the present invention may consist of a one-step method (one reagent system) or a multi-step method (multiple reagent system).

  When the enzyme activity measurement method in the biological sample of the present invention comprises a two-reagent system, when the first reagent and the second reagent are mixed in the ratio of the addition amount at the time of measurement, Mixing the first reagent, the second reagent, and the biological sample of the enzyme activity measurement reagent containing zinc ions in the first reagent and / or the second reagent so that the zinc ion concentration is 8 to 1200 ppb, and a trace amount of zinc ions In the presence of the enzyme, the enzyme reaction is carried out, the absorbance in the wavelength region of 400 to 500 nm of the substance produced by the reaction of the enzyme with the substrate or the substance derived from the product is measured, and the enzyme in the biological sample is determined from the measured value. Calculate activity.

  As mentioned above, the ratio when the amount of sample added is divided by the amount of enzyme activity measuring reagent is usually within the range of 1 to 5%, and the amount of sample added is almost negligible. Therefore, the concentration of a minute amount of zinc ions when the enzyme reaction is performed is almost the same as 8-1200 ppb (preferably 8-1000 ppb, more preferably 8-800 ppb, particularly preferably 8-600 ppb). .

In the method for measuring enzyme activity in a biological sample of the present invention, the amount of change per minute in the absorbance in the wavelength region of 400 to 500 nm is determined by the production rate of a product produced by reacting an enzyme with a substrate or a substance derived from this product. Any method can be used as long as it is based on the measurement principle calculated by measuring the absorbance in the wavelength range of 400 to 500 nm or the amount of the product produced by the reaction of the enzyme with the substrate or the substance derived from the product. Can also be used. For example, it can be applied to a reaction rate method (rate method) or an end point method (end point method).
In such a measuring method, the enzyme activity can be measured by a method known per se or a method using an automatic analyzer.

For example, glycerophosphate, 4-nitrophenyl phosphate or the like is used as a measurement substrate for alkaline phosphatase; 4-nitrophenyl-α-D-maltoheptaoside, 4 is used as a measurement substrate for α-amylase. , 6-benzylidene-4-nitrophenyl-.alpha.-D-maltoheptaoside, ^{4 5}
-O-β-D-galactopyranosyl-4-nitrophenyl-α-D-maltopentaoside, 2-chloro-4-nitrophenyl-β-D-maltopentaoside, etc. are used; Butyrylthiocholine or the like is used as a measurement substrate; L-γ-glutamyl-4-nitroanilide, L-γ-glutamyl-3-carboxy-4-nitro is used as a measurement substrate for γ-glutamyltranspeptidase. Anilide or the like is used; L-Leucyl-4-nitroanilide or the like is used as a measurement substrate for leucine aminopeptidase; S-benzyl-L-cysteine- is used as a measurement substrate for L-cystine aminotranspeptidase. 4-Nitroanilide or the like is used.
It has an absorption maximum at 400 to 500 nm, such as 4-nitrophenol, 2-chloro-4-nitrophenol, 4-nitroaniline or 3-carboxy-4-nitroaniline released by the action of enzymes on these substrates. The absorbance of the substance in the wavelength range of 400 to 500 nm is measured over time, and the enzyme activity is calculated from the increase in absorbance.

Alternatively, a substance such as thiocholine released by the action of an enzyme on these substrates reacts with the SH group to quantitatively change the absorbance in the wavelength range of 400 to 500 nm. (2-nitrobenzoic acid)] and the like are added and reacted to measure the absorbance in the wavelength region of 400 to 500 nm over time of the generated substance having an absorption maximum at 400 to 500 nm. From the increase in the absorbance, the enzyme is measured. Calculate activity.
In this case, according to the present invention, it is possible to suppress errors caused by the decomposition of hemoglobin in the biological sample.

[7] Method for avoiding the influence of hemoglobin The method for avoiding the influence of hemoglobin in the present invention is a method for measuring enzyme activity in a biological sample, in which measurement is performed in the presence of zinc ions at a concentration of 8 to 1200 ppb. Is due to.
In this method for measuring enzyme activity in a biological sample, even if hemoglobin is mixed in the biological sample due to hemolysis, the measurement is performed in the presence of zinc ions having a concentration of 8 to 1200 ppb. An error in the measurement value due to hemoglobin can be avoided, and a highly accurate measurement value of enzyme activity can be obtained.
In addition, the components of the reagent, the biological sample, the conditions, and the like in carrying out the method for avoiding the influence of hemoglobin generated by hemolysis in the present invention are as described above.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by these Examples.

[Example 1]
(Confirmation of effects of avoiding the effects of hemoglobin according to the present invention)
A hemoglobin obtained by preparing an alkaline phosphatase (ALP) activity measuring reagent based on the recommended method of the Japanese Society for Clinical Chemistry (JSCC), comprising a first reagent and a second reagent, and containing zinc ions in the first reagent Confirmed the effect of avoiding the effects of.

1. Reagent preparation

(1) Preparation of ALP first reagent The following reagent components are dissolved in pure water so as to have the concentrations indicated, and the pH is adjusted to 10.1 (20 ° C.) with 2N hydrochloric acid, so that the zinc sulfate concentration differs. Twelve kinds of ALP first reagents were prepared.

2-ethylaminoethanol (buffer) 1.01M
Magnesium chloride (hexahydrate) 0.505 mM
Zinc sulfate (7-hydrate) 0 ppb, 10 ppb, 50 ppb, 100 ppb, 150 ppb, 200 ppb, 400 ppb, 600 ppb, 800 ppb, 1000 ppb, 1200 ppb, or 1400 ppb

(2) Preparation of ALP Second Reagent The following reagent components are dissolved in pure water so as to have the respective concentrations described above, pH is adjusted to 10.2 (20 ° C.) with 2N hydrochloric acid, and ALP second reagent is prepared. Prepared.

2-ethylaminoethanol (buffer) 1.01M
4-nitrophenyl phosphate disodium salt 75.75 mM
Magnesium chloride (hexahydrate) 0.505 mM

2. Sample preparation 9 volumes of a human serum sample and 1 volume of an aqueous solution containing no hemoglobin, an interference check A plus Hb (manufactured by Sysmex) or an aqueous solution having a hemoglobin concentration of 5000 mg / dL prepared by hemolyzing human erythrocytes, respectively. Mixed to prepare serum samples with hemoglobin concentrations of 0, 100, 200, 300, 400, or 500 mg / dL, respectively.

3. Measurement of alkaline phosphatase activity in human serum samples Measurement of alkaline phosphatase activity in human serum samples is carried out using a 7180 automatic analyzer manufactured by Hitachi, Ltd. ALP first reagent is used as the first reagent in 2.5 μL of human serum sample. After adding 160 μL and reacting at 37 ° C. for 5 minutes, 40 μL of ALP second reagent is added as the second reagent to form a mixed solution (final reaction solution), and the reaction is performed at 37 ° C., and the second reagent is added at 1 minute and 10 seconds The alkaline phosphatase activity value was calculated from the rate of increase in absorbance at the primary wavelength of 405 nm and the secondary wavelength of 505 nm from the (21 point) to the 5th minute (34 points).

  In the present invention, calibration and calibration were performed using an enzyme calibrator (Aalto EC; manufactured by Sinotest), and the alkaline phosphatase activity value was calculated.

In addition, the concentration of zinc sulfate in the measurement reagent after mixing the above-mentioned 12 kinds of ALP first reagent and the above ALP second reagent in the above-mentioned addition amount is as follows.
0 ppb, 8 ppb, 40 ppb, 80 ppb, 120 ppb, 160 ppb, 320 ppb, 480 ppb, 640 ppb, 800 ppb, 960 ppb, or 1120 ppb

4). Measurement results Table 1 shows the measurement results.
In Table 1, the values in parentheses are the hemoglobin concentrations of 100 mg / dL, 200 mg / dL, and 300 mg when the ALP activity measurement value of a serum sample with a hemoglobin concentration of 0 mg / dL is 100% in each of the 12 types of reagents. ALP activity measured value of serum sample of / dL, 400 mg / dL, or 500 mg / dL is expressed as a relative ratio (%). The zinc sulfate concentration indicates the zinc sulfate concentration (ppb) in the measurement reagent after mixing.

5. Discussion As is apparent from Table 1, it can be seen that when the reagent does not contain zinc sulfate (zinc ion), a negative error occurs in the measured ALP activity due to the influence of hemoglobin in the sample. . Furthermore, it can be seen that as the hemoglobin concentration in the sample increases, the ALP activity measurement value decreases and the degree of negative error increases.
On the other hand, when zinc sulfate (zinc ion) is contained in the first reagent, the negative error of the measured value is reduced and improved in any case of the zinc sulfate concentration in the measurement reagent after mixing. I understand that. That is, it can be seen that the error caused by the influence of hemoglobin can be suppressed at the zinc sulfate concentration in the measurement reagent after mixing at concentrations of 8 ppb or more and 1200 ppb or less.

As a result, the reagent for measuring enzyme activity in a biological sample contains a small amount of zinc ions of 8 to 1200 ppb, so that an error in the measured value due to hemoglobin can be avoided even in a hemolyzed sample. It was confirmed that a high enzyme activity value can be obtained.
Therefore, it was confirmed that the enzyme activity measuring reagent and measuring method of the present invention can obtain an accurate enzyme activity value without causing an error even in a hemolyzed sample.

[Example 2]
(Confirmation of effects of avoiding the effects of hemoglobin according to the present invention)
A hemoglobin obtained by preparing an alkaline phosphatase (ALP) activity measuring reagent based on the recommended method of the Japanese Society for Clinical Chemistry (JSCC), comprising a first reagent and a second reagent, and containing zinc ions in the first reagent Confirmed the effect of avoiding the effects of.

1. Reagent preparation

(1) Preparation of ALP first reagent The following reagent components are dissolved in pure water so as to have the concentrations indicated, and the pH is adjusted to 10.1 (20 ° C.) with 2N hydrochloric acid, so that the zinc sulfate concentration differs. Seven types of ALP first reagents were prepared.

2-ethylaminoethanol (buffer) 1.01M
Magnesium chloride (hexahydrate) 0.505 mM
Zinc sulfate (7-hydrate) 0 ppb, 100 ppb, 150 ppb, 200 ppb, 400 ppb, 600 ppb, or 800 ppb

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

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

3. Measurement of Alkaline Phosphatase Activity in Human Serum Sample Each of the two samples was measured in the same manner as in Example 1 3 using the seven types of ALP first reagent and ALP second reagent prepared in 1 above, and hemoglobin was obtained. Confirmed the effect of avoiding the effects of.

The concentration of zinc sulfate in the measurement reagent after mixing the above seven types of ALP first reagent and the above ALP second reagent in the above addition amounts is as follows.
0 ppb, 80 ppb, 120 ppb, 160 ppb, 320 ppb, 480 ppb, or 640 ppb

4). Measurement results Table 2 shows the measurement results.
In Table 2, the values in parentheses indicate hemoglobin concentrations of 100 mg / dL, 200 mg / dL, and 300 mg when the ALP activity measurement value of a serum sample having a hemoglobin concentration of 0 mg / dL is 100% in each of the seven types of reagents. ALP activity measured value of serum sample of / dL, 400 mg / dL, or 500 mg / dL is expressed as a relative ratio (%). The zinc sulfate concentration indicates the zinc sulfate concentration (ppb) in the measurement reagent after mixing.

5. Discussion As is apparent from Table 2, when the reagent does not contain zinc sulfate (zinc ion), it is understood that a negative error occurs in the measured ALP activity due to the influence of hemoglobin in the sample. . Furthermore, it can be seen that as the hemoglobin concentration in the sample increases, the ALP activity measurement value decreases and the degree of negative error increases.
On the other hand, when zinc sulfate (zinc ion) is contained in the first reagent, the negative error of the measured value is reduced and improved in any case of the zinc sulfate concentration in the measurement reagent after mixing. I understand that. That is, it can be seen that errors caused by the influence of hemoglobin can be suppressed.

As a result, the reagent for measuring enzyme activity in biological samples can contain a small amount of zinc ions, so that even hemolyzed samples can avoid errors in measured values due to hemoglobin, resulting in highly accurate enzyme activity. It was confirmed that the value could be obtained.
Therefore, it was confirmed that the enzyme activity measuring reagent and measuring method of the present invention can obtain an accurate enzyme activity value without causing an error even in a hemolyzed sample.

Example 3
(Confirmation of effects of avoiding the effects of hemoglobin according to the present invention)
An alkaline phosphatase (ALP) activity measuring reagent that is composed of a first reagent and a second reagent and is compliant with the Japanese Society for Clinical Chemistry (JSCC) recommended method is prepared, and zinc ions are contained in the first reagent and the second reagent. The effect of avoiding the effects of hemoglobin was confirmed.

1. Reagent preparation

(1) Preparation of ALP first reagent The ALP first reagent prepared in (1) of Example 2 was used as the ALP first reagent.

(2) Preparation of ALP second reagent a The ALP second reagent prepared in (2) of Example 2 was used as the second reagent a.

(3) Preparation of ALP second reagent b The components and concentrations are the same as those of the ALP second reagent prepared in (2) of Example 2 except that it contains 600 ppb of zinc sulfate (7-hydrate). An ALP second reagent b was prepared.

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

3. Measurement of Alkaline Phosphatase Activity in Human Serum Sample For each of the two samples, ALP first reagent and ALP second reagent a containing no zinc sulfate (0 ppb) prepared in 1 above, and seven types of ALP first reagents and Measurement was performed with the ALP second reagent b in the same manner as in Example 3 described above, and the effect of avoiding the influence of hemoglobin was confirmed.

In addition, the concentration of zinc sulfate in the measurement reagent after mixing the ALP first reagent and the ALP second reagent a not containing zinc sulfate (0 ppb) with the above addition amount is 0 ppb.
In addition, the concentration of zinc sulfate in the measurement reagent after mixing the above seven types of ALP first reagent and the above ALP second reagent b with the above addition amounts is as follows.
120 ppb, 200 ppb, 240 ppb, 280 ppb, 440 ppb, 600 ppb, or 760 ppb

4). Measurement results Table 3 shows the measurement results.
In addition, the numerical values shown in Table 3 are hemoglobin concentrations of 100 mg / dL, 200 mg / dL, 300 mg / dL, and 400 mg when the ALP activity measurement value of a serum sample having a hemoglobin concentration of 0 mg / dL is defined as 100%. ALP activity measured value of serum sample of / dL or 500 mg / dL is expressed as a relative ratio (%). The zinc sulfate concentration indicates the zinc sulfate concentration (ppb) in the measurement reagent after mixing.

5. Discussion As is apparent from Table 3, when the reagent does not contain zinc sulfate (zinc ion), it is understood that a negative error occurs in the measured ALP activity due to the influence of hemoglobin in the sample. . Furthermore, it can be seen that as the hemoglobin concentration in the sample increases, the ALP activity measurement value decreases and the degree of negative error increases.
On the other hand, when zinc sulfate (zinc ion) is contained in the first reagent and the second reagent, the negative error of the measured value is reduced regardless of the zinc sulfate concentration in the measurement reagent after mixing. It can be seen that it has been improved. That is, it can be seen that errors caused by the influence of hemoglobin can be suppressed.

As a result, the reagent for measuring enzyme activity in biological samples can contain a small amount of zinc ions, so that even hemolyzed samples can avoid errors in measured values due to hemoglobin, resulting in highly accurate enzyme activity. It was confirmed that the value could be obtained.
Therefore, it was confirmed that the enzyme activity measuring reagent and measuring method of the present invention can obtain an accurate enzyme activity value without causing an error even in a hemolyzed sample.

Example 4
(Confirmation of effects of avoiding the effects of hemoglobin according to the present invention)
A hemoglobin in which an alkaline phosphatase (ALP) activity measuring reagent based on the Japanese Society for Clinical Chemistry (JSCC) recommended method, which is composed of a first reagent and a second reagent, is prepared and zinc ion is contained in the second reagent Confirmed the effect of avoiding the effects of.

1. Reagent preparation

(1) Preparation of ALP first reagent The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to 8.0 (20 ° C) with 1N hydrochloric acid. Prepared.

4-Nitrophenyl phosphate disodium salt 18.94 mM
Magnesium chloride (hexahydrate) 0.505 mM

(2) Preparation of ALP Second Reagent The following reagent components are dissolved in pure water so that each concentration is as described, and the pH is adjusted to 10.2 (20 ° C.) with 2N hydrochloric acid, so that the zinc sulfate concentration is different. Seven types of ALP second reagents were prepared.

2-ethylaminoethanol (buffer solution) 5.05 mM
Magnesium chloride (hexahydrate) 0.505 mM
Zinc sulfate (7-hydrate) 0 ppb, 1000 ppb, 2000 ppb, 3000 ppb, 4000 ppb, 5000 ppb, or 6000 ppb

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

3. Measurement of Alkaline Phosphatase Activity in Human Serum Sample Each of the above two samples was measured in the same manner as in Example 1 3 with the ALP first reagent and the seven types of ALP second reagents prepared in 1 above, and hemoglobin was obtained. Confirmed the effect of avoiding the effects of.

In addition, the concentration of zinc sulfate in the measurement reagent after mixing the ALP first reagent and the seven types of ALP second reagents in the above addition amounts is as follows.
0 ppb, 200 ppb, 400 ppb, 600 ppb, 800 ppb, 1000 ppb, or 1200 ppb

4). Measurement results Table 4 shows the measurement results.
The numerical values shown in Table 4 are the hemoglobin concentrations of 100 mg / dL, 200 mg / dL, 300 mg / dL, and 400 mg when the ALP activity measurement value of a serum sample with a hemoglobin concentration of 0 mg / dL is 100%. ALP activity measured value of serum sample of / dL or 500 mg / dL is expressed as a relative ratio (%). The zinc sulfate concentration indicates the zinc sulfate concentration (ppb) in the measurement reagent after mixing.

5. Discussion As is apparent from Table 4, when the reagent does not contain zinc sulfate (zinc ion), it can be seen that there is a negative error in the measured ALP activity due to the influence of hemoglobin in the sample. . Furthermore, it can be seen that as the hemoglobin concentration in the sample increases, the ALP activity measurement value decreases and the degree of negative error increases.
On the other hand, when zinc sulfate (zinc ion) is contained in the second reagent, the negative error of the measured value is reduced and improved in any case of the zinc sulfate concentration in the measurement reagent after mixing. I understand that. That is, it can be seen that errors caused by the influence of hemoglobin can be suppressed.

As a result, the reagent for measuring enzyme activity in biological samples can contain a small amount of zinc ions, so that even hemolyzed samples can avoid errors in measured values due to hemoglobin, resulting in highly accurate enzyme activity. It was confirmed that the value could be obtained.
Therefore, it was confirmed that the enzyme activity measuring reagent and measuring method of the present invention can obtain an accurate enzyme activity value without causing an error even in a hemolyzed sample.

Claims (8)

A reagent for measuring alkaline phosphatase activity in a biological sample containing zinc ions at a concentration of 8-1200 ppb and using 2-ethylaminoethanol as a buffer. The reagent for measuring alkaline phosphatase activity in a biological sample according to claim 1, wherein the reagent for measuring alkaline phosphatase activity measures absorbance in a wavelength region of 400 to 500 nm. The alkaline phosphatase activity measuring reagent is composed of two reagents, a first reagent and a second reagent, and zinc ions are contained in at least either the first reagent or the second reagent. The reagent for measuring alkaline phosphatase activity in the biological sample as described. The reagent for measuring alkaline phosphatase activity in a biological sample according to claim 1, wherein zinc ions are contained as an influence avoidance agent for hemoglobin. An alkaline phosphatase activity measuring reagent containing zinc ions at a concentration of 8 to 1200 ppb and using 2-ethylaminoethanol as a buffer is mixed with a biological sample, and an enzyme reaction is performed in the presence of zinc ions, and 400 to 500 nm. A method for measuring alkaline phosphatase activity in a biological sample, wherein the absorbance in the wavelength region of is measured. The alkaline phosphatase activity measuring reagent is composed of two reagents, a first reagent and a second reagent, and zinc ions are contained in at least one of the first reagent and the second reagent. A method for measuring alkaline phosphatase activity in a biological sample. The method for measuring alkaline phosphatase activity in a biological sample according to claim 5 or 6, wherein zinc ions are contained as an agent for avoiding the influence of hemoglobin. An agent for avoiding the influence of hemoglobin at the time of measuring alkaline phosphatase activity in a biological sample comprising 2-ethylaminoethanol as a buffer, comprising zinc ions at a concentration of 8 to 1200 ppb.